Stimulation of Nitric Oxide Production in Macrophages by Babesia bovis

Gamma interferon (IFN-γ)-activated macrophages are believed to play a key role in resistance to Babesia bovis through parasite suppression by macrophage secretory products. However, relatively little is known about interactions between this intraerythrocytic parasite and the macrophages of its bovine host. In this study, we examined the in vitro effect of intact and fractionated B. bovis merozoites on bovine macrophage nitric oxide (NO) production. In the presence of IFN-γ, B. bovis merozoites stimulated NO production, as indicated by the presence of increased l-arginine-dependent nitrite (NO₂⁻) levels in culture supernatants of macrophages isolated from several cattle. The merozoite crude membrane (CM) fraction stimulated greater production of NO, in a dose-dependent manner, than did the merozoite homogenate or the soluble, cytosolic high-speed supernatant fraction. Stimulation of NO production by CM was enhanced by as little as 1 U of IFN-γ per ml of culture medium. Upregulation of inducible NO synthase mRNA in bovine macrophages by either B. bovis-parasitized erythrocytes and IFN-γ or CM was also observed. B. bovis-specific T-helper lymphocyte culture supernatants, all of which contained IFN-γ, were also found to induce l-arginine-dependent NO₂⁻ production. Supernatants that induced the highest levels of NO also contained biologically active TNF. These results show that B. bovis merozoites and antigen-stimulated B. bovis-immune T cells can induce the production of NO, a molecule implicated in both protection and pathologic changes associated with hemoprotozoan parasite infections.Bovine babesiosis is an economically important tick-borne disease of cattle that is caused by intraerythrocytic apicomplexan parasites of the genus Babesia. Babesia bovis-infected erythrocytes undergo sequestration by attachment to capillary endothelium in a manner reminiscent of the most severe form of human malaria, caused by Plasmodium falciparum; this results in organ damage, cerebral dysfunction, and pulmonary edema (58). It has been hypothesized that the severe organ abnormalities that occur during acute B. bovis infection, similar to those observed during experimental malaria, are mediated in part by inflammatory cytokines, including gamma interferon (IFN-γ) and tumor necrosis factor (TNF), and nitric oxide (NO) (58).Although activation of macrophages could lead to immunopathological consequences, macrophages are also believed to be important for immunity to B. bovis and other intraerythrocytic parasites via removal of parasitized erythrocytes by phagocytosis and as antigen-presenting cells (APC) for T-helper (Th) lymphocytes. Furthermore, macrophage secretory products have been shown to inhibit the growth of P. falciparum and B. bovis in vitro (24, 34). When generated by chemical donors or activated macrophages in vitro, NO and its reactive nitrogen intermediate derivatives were shown to inhibit intracellular parasites including Leishmania major, P. falciparum, and B. bovis (22, 23, 27, 40, 53, 54).Studies in mice with B. microti, P. yoelii, P. vinckei, and P. chabaudi demonstrated that IFN-γ, TNF-α, and TNF-β are important components of immunity to these parasites (3, 15, 32, 44, 47). IFN-γ facilitates the phagocytosis of P. falciparum-infected erythrocytes by human macrophages (33, 35). In addition, parasite-specific, IFN-γ-producing human CD4⁺ Th-cell clones inhibited the growth of P. falciparum in the presence of adherent peripheral blood mononuclear cells (PBMC) in vitro (18, 37). The protective role of TNF-α appears to depend on the timing of its appearance, which may partially explain the paradoxical roles of TNF in protection and immunopathology. In resistant C57BL/6 mice, TNF-α appeared early during P. chabaudi infection, whereas in susceptible A/J mice, high levels of TNF-α were observed only late in infection, just preceding death (25).Experiments performed during the past decade showed that malarial parasites and secreted toxins induced TNF-α in human and murine macrophages and inducible NO synthase (iNOS) in murine macrophages. Macrophages exposed in vitro to P. yoelii, P. berghei, or P. falciparum produced TNF-α, which was enhanced by IFN-γ (4, 36, 50, 51). Similarly, P. falciparum extract, in the presence of IFN-γ, stimulated murine macrophages to produce both TNF-α and NO (31, 41). However, the interpretation of these results has been questioned by the recent discovery that many continuously cultured strains of P. falciparum are contaminated with Mycoplasma species (56). Mycoplasma organisms induce TNF-α and other inflammatory mediators in murine, human, and bovine macrophages (28–30, 56), and experiments with Mycoplasma-free P. falciparum are being repeated to verify that the induction of inflammatory cytokines and NO was due to the parasite itself (43).Several observations support the hypothesis that IFN-γ produced by effector Th cells plays a key role in protection against babesiosis (10). In cattle, IFN-γ regulates B-cell synthesis of the opsonizing immunoglobulin G2 subclass (17). Parasite-specific Th-cell clones isolated from B. bovis-immune cattle produce IFN-γ and TNF (11, 14). Bovine macrophages have upregulated expression of iNOS when activated by IFN-γ in the presence of bacterial LPS (2) or TNF-α (20). Thus, the induction of macrophage iNOS by either B. bovis extracts or antigen-activated T cells would be indicative of macrophage activation and a potential babesiacidal effector mechanism. Conversely, overproduction of NO could also contribute to the pathologic changes associated with infection, including cerebral babesiosis.This study was undertaken to determine if bovine macrophages produce NO when exposed to B. bovis in the presence or absence of IFN-γ. Potential Mycoplasma contamination of B. bovis cultures was ruled out by a PCR-based assay. We report, for the first time, that bovine macrophages produce NO following in vitro exposure to B. bovis merozoites or a membrane-enriched fraction. The effect of different concentrations of either IFN-γ or B. bovis on NO production was also examined. Furthermore, the functional relevance of B. bovis-specific Th cells that produce IFN-γ and TNF was evaluated by the determining the ability of Th-cell supernatants to stimulate NO production. Induction of iNOS was confirmed by demonstrating reduced nitrite (NO₂⁻) levels in the presence of N^G-monomethyl-l-arginine (l-NMMA) and enhanced levels of iNOS steady-state mRNA in macrophages cultured with B. bovis.     

Laminin α4-Null Mutant Mice Develop Chronic Kidney Disease with Persistent Overexpression of Platelet-Derived Growth Factor

Each extracellular matrix compartment in the kidney has a unique composition, with regional specificity in the expression of various laminin isoforms. Although null mutations in the majority of laminin chains lead to specific developmental abnormalities in the kidney, Lama4−/− mice have progressive glomerular and tubulointerstitial fibrosis. These mice have a significant increase in expression of platelet-derived growth factor (PDGF)-BB, PDGF-DD, and PDGF receptor β in association with immature glomerular and peritubular capillaries. In addition, mesangial cell exposure to α4-containing laminins, but not other isoforms, results in down-regulation of PDGF receptor mRNA and protein, suggesting a direct effect of LN411/LN421 on vessel maturation. Given the known role of overexpression of PDGF-BB and PDGF-DD on glomerular and tubulointerstitial fibrosis, these data suggest that failure of laminin α4-mediated down-regulation of PDGF activity contributes to the progressive renal lesions in this animal model. Given the recent demonstration that individuals with laminin α4 mutations develop cardiomyopathy, these findings may be relevant to kidney disease in humans.Laminin (LN) is a large, heterotrimeric, cruciform molecule composed of α, β, and γ subunits.¹ Five distinct α (LAMA1-5), 3 β (LAMB1-3), and 3 γ (LAMC1-3) chains^1,2 variably assemble to create distinct isoforms³ that are temporally and spatially regulated, and each conveys a variety of biological functions.^{4,5,6,7,8,9,10,11} The LNα4-containing isoforms, LN411 (α4β1γ1) and LN421 (α4β2γ1), are abundant in microvessels. Studies of LNα4-deficient mutant mice (lama4−/−) reveal that although α4-LNs are not required for blood vessel formation, they play important roles in blood vessel maturation, and in stabilization of vessels that form with injury, inflammation and tumor growth.^7,12,13 In vitro studies indicate that α4LNs directly regulate endothelial cell proliferation and inhibit apoptosis.¹⁴ α4-LNs are produced by endothelial cells in most microvessels; however, endothelial cells in the renal glomerulus do not express LNα4-containing isoforms.¹⁵ Instead, LN411 and LN421 at the endothelial-mesangial interface are produced by the mesangial cells (MCs).¹⁵ Platelet-derived growth factor (PDGF) is the primary growth factor responsible for MC proliferation and migration during glomerulogenesis,¹⁶ and we have shown that PDGF-induced MC migration requires LNα4.¹⁵ This function could not be replaced by LN111 or LN511/521.¹⁵ Together these observations suggested the possibility that deficiency of LNα4 might impair the ability of the kidney microvasculature to mature or be repaired in lama4−/− adult mice, resulting in kidney disease despite normal initial development.Previous reports have documented a spectrum of developmental defects and tissue maintenance defects in lama4−/− mice. Early postal-natal hemorrhage from birth-related trauma to fragile blood vessels occurs in lama4−/− mice; yet, by three-weeks of age, accumulation of LNα5 stabilizes vessels, although they remain dilated.¹² Vessel fragility recurs when new vessels form in response to injury.¹² The heart forms normally, but lama4−/− mice develop cardiomyopathy with time.¹⁷ Neurological dysfunction occurs in lama4−/− mice, through independent defects in organizing presynaptic specializations at neuromuscular synapses,¹⁸ and in the ability of developing Schwann cells to properly sort and myelinate.^19,20 This report details the characteristics of kidney abnormalities, including the development of glomerulosclerosis and tubulointerstitial fibrosis over time in lama4−/− mice.     

T-Cell Activation Leads to Reduced Collagen Maturation in Atherosclerotic Plaques of Apoe−/− Mice

Rupture of the collagenous, fibrous cap of an atherosclerotic plaque commonly causes thrombosis. Activated immune cells can secrete mediators that jeopardize the integrity of the fibrous cap. This study aimed to determine the relationship between T-cell-mediated inflammation and collagen turnover in a mouse model of experimental atherosclerosis. Both Apoe^−/− × CD4dnTβRII mice with defective transforming growth factor-β receptors in T cells (and hence released from tonic suppression of T-cell activation) and lesion size-matched Apoe^−/− mice were used. Picrosirius red staining showed a lower content of thick mature collagen fibers in lesions of Apoe^−/− × CD4dnTβRII mice, although both groups had similar levels of procollagen type I or III mRNA and total collagen content in lesions. Analysis of both gene expression and protein content showed a significant decrease of lysyl oxidase, the extracellular enzyme needed for collagen cross-linking, in aortas of Apoe^−/− − CD4dnTβRII mice. T-cell-driven inflammation provoked a selective and limited increase in the expression of proteinases that catabolize the extracellular matrix. Atheromata of Apoe^−/− − CD4dnTβRII mice had increased levels of matrix metalloproteinase-13 and cathepsin S mRNAs and of the active form of cathepsin S protein but no increase was detected in collagen fragmentation. Our results suggest that exaggerated T-cell-driven inflammation limits collagen maturation in the atherosclerotic plaque while having little effect on collagen degradation.A physical disruption of atherosclerotic plaques causes many acute thrombotic complications such as myocardial infarction and stroke.^1,2 Pathologists have identified a number of characteristics of vulnerable atherosclerotic plaques that have ruptured³ including large lipid cores and thin fibrous caps harboring activated macrophages and T cells.^1,4 The resistance of the atherosclerotic plaque to disruption depends in part on the integrity of its fibrous cap, which prevents contact between the highly thrombogenic lipid core and the circulating blood.^1,2 The fibrous cap is composed of smooth muscle cells (SMCs) and a collagen-rich extracellular matrix. The fibrillar collagens types I and III synthesized by SMCs primarily determine the tensile strength of the cap. Sites of plaque rupture characteristically display signs of inflammatory cell activation accompanied by dissolution of matrix.Inflammation may impair plaque stability because macrophages and mast cells release a set of collagen-degrading matrix metalloproteinases (MMPs) and cysteine proteases.^5,6,7 Additional possible mechanisms include inhibited expression of procollagen genes and death or reduced renewal of the collagen-producing SMC population, both phenomena promoted by T-cell-derived interferon (IFN)-γ.⁸ Several studies suggest that T cells may modulate plaque stability. Activated T cells accumulate in vulnerable plaques.⁴ Th1 cells, which dominate in plaques, secrete IFN-γ, a powerful inhibitor of endothelial and SMC proliferation.⁹ IFN-γ also inhibits differentiation and collagen gene expression in SMCs^10,11 and modulates expression of several MMPs and cathepsins.^12,13 In vivo treatment with IFN-γ increases atherosclerosis and transplant arteriosclerosis,^14,15 whereas targeted gene deletion in the IFN-γ receptor leads to reduced atherosclerosis in hypercholesterolemic mice.¹⁶ Mice with defective control of T-cell activity show modulation of atherogenesis: interleukin-10 targeted mice display increased plaque formation with reduced collagen accumulation,¹⁷ and Apoe^−/− mice lacking transforming growth factor (TGF)-β inhibition of T cells rapidly develop large, hyperinflammatory lesions.¹⁸ These data point to a proatherosclerotic and possibly destabilizing role for activated T cells.The present study aimed to assess the effect of inflammation on the fibrous component of the atherosclerotic plaque. We used Apoe^−/− × CD4dnTβRII mice, which lack functional TGF-β receptors on T cells. Therefore, uncontrolled T-cell activation leads to rampant inflammation and accelerated atherosclerosis in hypercholesterolemic animals.¹⁸ Therefore, Apoe^−/− × CD4dnTβRII mice provide an opportunity to study the effect of inflammation on atherosclerotic lesions. Our data show reduced enzyme-dependent collagen maturation in hyperinflamed lesions, whereas effects on procollagen expression and collagenolytic enzymes were modest. These results suggest a novel mechanism by which adaptive immunity can modulate plaque stability—impairment of collagen maturation by T-cell-dependent inflammation.     

The Cysteine-Cysteine Family of Chemokines RANTES,MIP-1α, and MIP-1β Induce Trypanocidal Activity in Human Macrophages via Nitric Oxide

This paper describes a new role for the cysteine-cysteine (CC) chemokines RANTES, MIP-1α, and MIP-1β on human macrophage function, which is the induction of nitric oxide (NO)-mediated trypanocidal activity. In a previous report, we showed that RANTES, MIP-1α and MIP-1β enhance Trypanosoma cruzi uptake and promote parasite killing by human macrophages (M. F. Lima, Y. Zhang, and F. Villalta, Cell. Mol. Biol. 43:1067–1076, 1997). Here we study the mechanism by which RANTES, MIP-1α, and MIP-1β activate human macrophages obtained from healthy individuals to kill T. cruzi. Treatment of human macrophages with different concentrations of RANTES, MIP-1α, and MIP-1β enhances T. cruzi trypomastigote phagocytosis in a dose peak response. The optimal response induced by the three CC chemokines is attained at 500 ng/ml. The macrophage trypanocidal activity induced by CC chemokines can be completely inhibited by l-N-monomethyl arginine (l-NMMA), a specific inhibitor of the l-arginine:NO pathway, but not by its d-enantiomer. Culture supernatants of chemokine-treated human macrophages contain increased NO₂⁻ levels, and NO₂⁻ production is also specifically inhibited by l-NMMA. The amount of NO₂⁻ induced by these chemokines in human macrophages is comparable to the amount of NO₂⁻ induced by gamma interferon. The killing of trypomastigotes by NO in cell-free medium is blocked by an NO antagonist or a NO scavenger. This data supports the hypothesis that the CC chemokines RANTES, MIP-1α, and MIP-1β activate human macrophages to kill T. cruzi via NO, which is an effective trypanocidal mechanism.As the role of the cysteine-cysteine (CC) family of chemokines in immunological processes continues to be defined, it is clear that their cellular influence and biological activities are broader and encompass more than simply chemoattraction. In fact, other functions beside chemotaxis, such as their participation in angiogenesis and neovascularization, resistance to human immunodeficiency virus type 1 (HIV-1), T-cell costimulatory activities (reviewed in references 2 and 46), and enhancement of NK-mediated cytolysis (45), have been reported. The mechanism by which CC chemokines activate human macrophages to exert cytotoxicity is unknown. In this study, we have explored the role of the CC chemokines RANTES, MIP1-α, and MIP-1β on human macrophage cytotoxic cell function against the human parasite Trypanosoma cruzi by examining their mechanism of trypanosome toxicity within macrophages.T. cruzi, the causative agent of Chagas’ disease, is an obligate intracellular pathogen of several cells including cells of the monocyte/macrophage lineage (4). This organism is now viewed as an emerging human pathogen of HIV-1-infected individuals, since it induces dramatic pathologic changes in the brain and results in earlier death when associated with HIV-1 infections (9, 39). The possible emergence of T. cruzi as an opportunistic infection of HIV-1-infected individuals in the United States has recently been considered (30). Inflammatory molecules have been postulated to play a role in the clearance of T. cruzi, since control of the acute phase of Chagas’ disease is critically dependent on cytokine-mediated macrophage activation. For instance, treatment of macrophages with gamma interferon (IFN-γ) (36), granulocyte-macrophage colony-stimulating factor (33, 37), or tumor necrosis factor alpha (TNF-α) (27) induces T. cruzi killing whereas transforming growth factor β (TGF-β) and interleukin-10 (IL-10) inhibit the trypanocidal action of IFN-γ-activated macrophages (14).Chemokines mediate inflammatory reactions (3, 40–42) and show potential leukocyte activation and/or chemotactic activity (11). CC chemokines act primarily on monocytes but also have been shown to act on basophils, eosinophils, lymphocytes including Th2 cells, astrocytes, dendritic cells, fibroblasts, and hematopoietic cells (reviewed in references 2 and 46). CC chemokines include MIP-1α and MIP-1β (macrophage inflammatory proteins 1α and 1β), RANTES (regulated upon activation, normal T expressed and secreted), MCP-1 through MCP-3 (7), I-309, Eotaxin, C10, HCC-1 (2, 46), and 6Ckine (16). The proinflammatory activities of CC chemokines overlap but are not identical (8); MIP-1α, MIP-1β, and RANTES bind to a common receptor, suggesting that there are structural similarities among them (8).Chemokines play important roles in immunopathogenesis and may selectively recruit cells into sites of antigenic challenge (7). They are active on lymphocytes and monocytes/macrophages upon binding to G-protein-coupled seven-transmembrane-domain surface receptors (13, 31). It was recently shown that HIV-1 entry into human macrophages is inhibited by MIP-1α, MIP-1β, and RANTES (6, 21) and that CCKR5, the RANTES, MIP-1α, and MIP-1β receptor, functions as a coreceptor for macrophage-tropic HIV-1 (1).The objective of this study was to investigate the microbicidal mechanism induced by these inflammatory secreted proteins in human macrophages infected with infective trypomastigote forms of T. cruzi. In this work, we describe a new role for the CC chemokines RANTES, MIP-1α, and MIP-1β on human macrophage function, which is the induction of microbicidal activity of these cells via NO.     

Gamma Interferon Treatment of Patients with Chronic Granulomatous Disease Is Associated with Augmented Production of Nitric Oxide by Polymorphonuclear Neutrophils

Treatment with gamma-interferon (IFN-γ) is associated with reduced frequency and severity of infections in chronic granulomatous disease (CGD), but the mechanism is unknown. Since the inducible nitric oxide (NO) synthase can be amplified by IFN-γ in murine macrophages, for example, we hypothesized that IFN-γ might modulate NO release from polymorphonuclear neutrophils (PMNs) in patients with CGD. Eight patients with CGD and eight healthy controls were studied. Each patient was given either 50 or 100 μg of IFN-γ per m² on two consecutive days. The production of NO from N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMNs was assessed as the N^G-monomethyl-l-arginine-inhibitable oxidation of oxyhemoglobin to methemoglobin in the presence of catalase and superoxide dismutase. Prior to IFN-γ treatment, the PMNs from CGD patients produced 372 ± 27 (mean ± standard error of the mean) pmol of NO/10⁶ PMNs at 45 min, while the control PMNs produced 343 ± 44 pmol. On day 1 after IFN-γ treatment, NO production increased to 132% ± 25% of that for controls, and on day 3 it reached 360% ± 37% (P < 0.001) of that for controls. On day 8, the values still remained higher, 280% ± 78% more than the control values. Likewise, the bactericidal capacity of PMNs increased on day 3. The present data show that IFN-γ treatment of CGD patients is associated with an increased production of NO from PMNs when activated by fMLP. Since these PMNs lack the capacity to produce superoxide anions, it is conceivable that this increase in NO release could be instrumental in augmenting host defense.Chronic granulomatous disease (CGD) is a rare X-linked or autosomal inherited disease characterized by recurrent life-threatening infections (27). The basic defect is an inability of phagocytic cells to produce superoxide anions and hydrogen peroxide, as a result of a defect in one of the subcomponents of the NADPH oxidase in these cells. In patients with the X-linked form of CGD, cells lack a membrane-associated part of the oxidase gp91^phox protein (27). Patients with the autosomal recessive form of CGD fail to demonstrate the presence of either one of two cytosolic factors, p47^phox and p67^phox, or the membrane-bound p22^phox (27).A multicenter study showed that recombinant human gamma interferon (IFN-γ) administered subcutaneously (s.c.) three times a week significantly reduced serious infections (17). This regimen has therefore been recommended since 1991 as prophylaxis against infections in patients with CGD. However, the mechanisms of action of IFN-γ in CGD are poorly understood. Although some early reports on variant forms of CGD showed a partially restored oxidative metabolism in cells of CGD patients after IFN-γ treatment (10, 11), studies of more common CGD phenotypes could not corroborate that observation (17, 25, 28, 30).Human polymorphonuclear neutrophils (PMNs) produce and release nitric oxide (NO) spontaneously (31) or following activation (21); both inducible and constitutive isoforms of NO synthase (NOS) have been purified from human PMNs (6, 29). Several lines of evidence imply that this function is of importance for host defense (22), possibly in CGD and furthermore in the inflammatory response as reviewed previously (14, 24). NO has been shown to possess cytotoxic (5) and bactericidal actions, particularly against intracellular pathogens (4, 13). In healthy individuals, NO released from PMNs reacts rapidly with superoxide anion-forming peroxynitrite (3). However, in CGD this reaction is not possible, and thus, NO by itself may be of a relatively larger importance. In murine systems, IFN-γ is a potent inducer of NOS (24). Inducible NOS activity in vivo has been demonstrated in PMNs from the urine of patients with urinary tract infections (29), a situation where PMNs are exposed to not only bacterial products, such as chemotactic oligopeptides, but also bacteria and a considerable presence of cytokines. Also, PMNs from CGD patients (herein referred to as CGD PMNs) have been shown to produce NO in vitro (7). Thus, we asked if the mechanism for improved host defense in CGD induced by IFN-γ could be associated with effects on PMN NO release, activated by a synthetic analogue to naturally occurring bacterial oligopeptides, and compared these data with those of a simultaneously run PMN microbicidal assay.     

Myotendinous Junction Defects and Reduced Force Transmission in Mice that Lack α7 Integrin and Utrophin

The α7β1 integrin, dystrophin, and utrophin glycoprotein complexes are the major laminin receptors in skeletal muscle. Loss of dystrophin causes Duchenne muscular dystrophy, a lethal muscle wasting disease. Duchenne muscular dystrophy-affected muscle exhibits increased expression of α7β1 integrin and utrophin, which suggests that these laminin binding complexes may act as surrogates in the absence of dystrophin. Indeed, mice that lack dystrophin and α7 integrin (mdx/α7^−/−), or dystrophin and utrophin (mdx/utr^−/−), exhibit severe muscle pathology and die prematurely. To explore the contribution of the α7β1 integrin and utrophin to muscle integrity and function, we generated mice lacking both α7 integrin and utrophin. Surprisingly, mice that lack both α7 integrin and utrophin (α7/utr^−/−) were viable and fertile. However, these mice had partial embryonic lethality and mild muscle pathology, similar to α7 integrin-deficient mice. Dystrophin levels were increased 1.4-fold in α7/utr^−/− skeletal muscle and were enriched at neuromuscular junctions. Ultrastructural analysis revealed abnormal myotendinous junctions, and functional tests showed a ninefold reduction in endurance and 1.6-fold decrease in muscle strength in these mice. The α7/utr^−/− mouse, therefore, demonstrates the critical roles of α7 integrin and utrophin in maintaining myotendinous junction structure and enabling force transmission during muscle contraction. Together, these results indicate that the α7β1 integrin, dystrophin, and utrophin complexes act in a concerted manner to maintain the structural and functional integrity of skeletal muscle.Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease that affects 1 in every 3500 live male births. Patients with DMD have impaired mobility, are restricted to a wheelchair by their teens, and die from cardiopulmonary failure in their early twenties.^1,2 Currently, there is no cure or effective treatment for this devastating disease. Mutations in the dystrophin gene resulting in loss of the dystrophin protein are the cause of disease in DMD patients and the mdx mouse model.^3,4,5,6,7The dystrophin glycoprotein complex links laminin in the extracellular matrix to the actin cytoskeleton. The N-terminal region of dystrophin interacts with cytoskeletal F-actin⁸ and the C-terminal region associates with the dystrophin-associated protein complex, which include α- and β-dystroglycan, α- and β-syntrophin, the sarcoglycans, and sarcospan.⁹ In DMD, the absence of dystrophin leads to disruption of the dystrophin glycoprotein complex, resulting in increased muscle fragility and altered cell signaling.⁹ Loss of this critical transmembrane linkage complex in DMD patients and mdx mice results in progressive muscle damage and weakness, inflammation, necrosis, and fibrosis. Lack of dystrophin also leads to abnormalities at myotendinous and neuromuscular junctions (MTJ and NMJ), which further contribute to skeletal muscle damage.^{10,11,12,13,14,15,16,17} In addition, defective muscle repair in DMD patients eventually results in muscle degeneration exceeding the rate of regeneration.¹⁸ Overall, dystrophin is critical for muscle function, structure, and stability, and its absence results in progressive muscle wasting and severe muscular dystrophy. In the absence of dystrophin two additional laminin-binding receptors, the α7β1 integrin and utrophin, are up-regulated in the skeletal muscle of DMD patients and mdx mice, which may compensate for the loss of the dystrophin glycoprotein complex.^19,20,21The α7β1 integrin is a heterodimeric laminin receptor involved in bidirectional cell signaling and is localized at junctional and extrajunctional sites in skeletal muscle.^22,23 At least six α7 integrin isoforms produced by developmentally regulated RNA splicing are expressed in skeletal muscle.²⁴ Mutations in the α7 integrin gene (ITGA7) cause myopathy in humans.²⁵ Mice lacking the α7 integrin develop myopathy, exhibit vascular smooth muscle defects and have altered extracellular matrix deposition.^{26,27,28,29,30} The observation that the α7β1 integrin is elevated in the muscle of DMD patients and mdx mice led to the hypothesis that the α7β1 integrin may compensate for the loss of dystrophin.¹⁹ Enhanced expression of the α7 integrin in the skeletal muscle of severely dystrophic mice reduced muscle pathology and increased lifespan by threefold.^10,11 In contrast, loss of both dystrophin and α7 integrin in mice results in severe muscular dystrophy and premature death by 4 weeks of age.^28,31 The α7β1 integrin is therefore a major modifier of disease progression in DMD.The utrophin glycoprotein complex is a third major laminin receptor in skeletal muscle. Utrophin has significant sequence homology to dystrophin.^32,33 In normal adult muscle utrophin is restricted to neuromuscular and myotendinous junctions.³⁴ During development or in damaged or diseased muscle, utrophin expression is increased and becomes localized at extrajunctional sites.^35,36 Utrophin interacts with the same proteins as dystrophin, but binds to actin filaments at different sites.³⁷ In mice, loss of utrophin results in a mild form of myasthenia with reduced sarcolemmal folding at the postsynaptic membrane of the neuromuscular junction.^12,15 Transgenic overexpression of utrophin has been shown to rescue mdx mice.³⁸ Mice that lack both dystrophin and utrophin exhibit severe muscular dystrophy and die by 14 weeks of age.^13,14 Thus, utrophin is also a major laminin receptor that modifies disease progression in DMD.To understand the functional overlap between the α7β1 integrin and utrophin in skeletal muscle, we produced mice that lack both α7 integrin and utrophin (α7/utr^−/−). Since both complexes are highly enriched at the MTJ and NMJ, we hypothesized that α7/utr^−/− mice may have severe abnormalities at these critical junctional sites. Our study demonstrates α7/utr^−/− mice exhibit partial embryonic lethality comparable with that observed in α7^−/− mice. Dystrophin is increased in these animals and enriched at the NMJ but not the MTJ. α7/utr^−/− mice display ultrastructural defects in their MTJ and compromised force transmission. Together, these results indicate that the α7β1 integrin, dystrophin and utrophin laminin binding complexes provide continuity between laminin in the extracellular matrix and the cell cytoskeleton, which are necessary for the normal structural and functional properties of skeletal muscle.     

In Vivo Regulation of Replicative Legionella pneumophila Lung Infection by Endogenous Interleukin-12

The in vivo role of endogenous interleukin 12 (IL-12) in modulating intrapulmonary growth of Legionella pneumophila was assessed by using a murine model of replicative L. pneumophila lung infection. Intratracheal inoculation of A/J mice with virulent bacteria (10⁶ L. pneumophila cells per mouse) resulted in induction of IL-12, which preceded clearance of the bacteria from the lung. Inhibition of endogenous IL-12 activity, via administration of IL-12 neutralizing antiserum, resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection (compared to untreated L. pneumophila-infected mice). Because IL-12 has previously been shown to modulate the expression of cytokines, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-10, which regulate L. pneumophila growth, immunomodulatory effects of endogenous IL-12 on intrapulmonary levels of these cytokines during replicative L. pneumophila lung infection were subsequently assessed. Results of these experiments demonstrated that TNF-α activity was significantly lower, while protein levels of IFN-γ and IL-10 in the lung were similar, in L. pneumophila-infected mice administered IL-12 antiserum, compared to similarly infected untreated mice. Together, these results demonstrate that IL-12 is critical for resolution of replicative L. pneumophila lung infection and suggest that regulation of intrapulmonary growth of L. pneumophila by endogenous IL-12 is mediated, at least in part, by TNF-α.

Legionella pneumophila, the causative agent of Legionnaires’ disease, is an intracellular pathogen of mononuclear phagocytic cells (MPCs) (37, 43, 45). Pulmonary infection usually develops following inhalation of L. pneumophila-contaminated water aerosols or microaspiration of contaminated water sources (9). Following inhalation, the bacteria invade and replicate in host MPCs, primarily in alveolar MPCs (34, 36, 37, 43, 45). Intracellular growth of L. pneumophila results in eventual lysis of infected MPCs, the release of bacterial progeny, and reinfection of additional pulmonary cells (34, 36). Severe lung damage, mediated by tissue-destructive substances likely derived from both damaged host cells and the bacteria, ensues (20, 21).Previous studies have demonstrated that resistance to primary replicative L. pneumophila lung infection is dependent on the induction of cellular immunity and is mediated in part by cytokines including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) (8, 12, 14, 15, 23, 27, 28, 35, 57). Growth of L. pneumophila within permissive MPCs requires iron. IFN-γ limits MPC iron, thereby converting the MPC intracellular environment from one that is permissive to one that is nonpermissive for L. pneumophila replication (14, 15). IFN-γ in combination with other cytokines including TNF-α facilitates elimination of L. pneumophila from infected MPCs, likely through the induction of effector molecules including nitric oxide (12). In contrast, other cytokines including interleukin 10 (IL-10) facilitate growth of L. pneumophila in permissive MPCs, due in part to IL-10-mediated inhibition of TNF-α secretion and IFN-γ-mediated MPC activation (46).IL-12 is a recently described cytokine with pleiotropic effects on T cells and natural killer (NK) cells which include (i) regulation of expression of cytokines including IFN-γ, TNF-α, and IL-10 by T cells and/or NK cells, (ii) induction of T-cell and/or NK cell proliferation and/or differentiation, and (iii) enhancement of NK cell and T-cell cytotoxic activity (4, 5, 19, 32, 33, 39, 44, 47, 48, 50, 56). While systemic administration of exogenous IL-12 has been demonstrated to increase host resistance to several intracellular pathogens, including Leishmania major, Toxoplasma gondii, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium, and Plasmodium chabaudi, in mice (26, 29, 33, 40, 51, 52, 55), the role of endogenous IL-12 in innate immunity to intracellular pathogens including L. pneumophila has not been thoroughly investigated. We have recently developed a model of replicative L. pneumophila lung infection in A/J mice inoculated intratracheally with virulent bacteria and have used this model system to identify immune responses which mediate host resistance to legionellosis (10–12). Using this murine model of Legionnaires’ disease, we assessed the biologic relevance and immunomodulatory role of endogenous IL-12 in innate immunity to replicative L. pneumophila lung infection.     

Interleukin-12 Is Required for Control of the Growth of Attenuated Aromatic-Compound-Dependent Salmonellae in BALB/c Mice: Role of Gamma Interferon and Macrophage Activation

The attenuated S. typhimurium SL3261 (aroA) strain causes mild infections in BALB/c mice. We were able to exacerbate the disease by administering anti-interleukin-12 (IL-12) antibodies, resulting in bacterial counts in the spleens and livers of anti-IL-12-treated mice that were 10- to 100-fold higher than the ones normally observed in premortem mice; yet the animals showed only mild signs of illness. Nevertheless, they eventually died of a slow, progressive disease. Mice infected with salmonellae become hypersusceptible to endotoxin. We found that IL-12 neutralization prevented the death of infected mice following subcutaneous injection of lipopolysaccharide. Granulomatous lesions developed in the spleens and livers of control animals, as opposed to a widespread infiltration of mononuclear cells seen in the organs of anti-IL-12-treated mice. In the latter (heavily infected), salmonellae were seen within mononuclear cells, indicating an impairment of the bactericidal or bacteriostatic ability of the phagocytes in the absence of biologically active IL-12. Gamma interferon (IFN-γ) levels were reduced in the sera and tissue homogenates from anti-IL-12-treated mice compared to those in control animals. Furthermore, fluorescence-activated cell sorter analysis on spleen cells showed that IL-12 neutralization impaired the upregulation of I-A^d/I-E^d antigens on macrophages from infected mice. Inducible nitric oxide synthase and IFN-γ mRNA production was down-regulated in anti-IL-12-treated mice, which also showed an increased production of IL-10 mRNA and a decrease in nitric oxide synthase activity in the tissues. Administration of recombinant IFN-γ to anti-IL-12-treated mice was able to restore host resistance, granuloma formation, and expression of major histocompatibility complex class II antigens in F4/80⁺ and CD11b⁺ spleen cells.Salmonella infections still pose a serious health hazard worldwide, affecting both humans and animals. Salmonella typhi, the agent of human typhoid fever, is not pathogenic for common laboratory animals. Therefore, natural resistance and acquired immunity to Salmonella are studied mainly in the mouse model by using host-adapted salmonellae which cause systemic infections believed to mimic the human disease.In mice, early bacterial growth in the reticuloendothelial system (RES) is controlled by the innate resistance Nramp (Ity) gene, which is expressed in macrophages (22). In lethal infections, salmonellae rapidly reach large numbers in the tissues and death occurs presumably by endotoxin poisoning when bacterial counts reach levels of ca. 10⁸ CFU per organ (30). In sublethal infections, survival requires a host response that suppresses the exponential growth of the organisms in the RES towards the end of the first week, resulting in a plateau phase (17, 25). The establishment of the plateau phase does not require functional T cells. In fact, nude (T-cell-deficient) mice and mice depleted of T cells by administration of anti-CD4 and anti-CD8 antibodies can still suppress Salmonella growth in infected tissues (17). A bone marrow-dependent influx of radiation-sensitive cells is required for the plateau phase and for the formation of granulomas rich in mononuclear cells (17, 32). Most of the salmonellae in the spleens and livers of the infected animals are localized within the phagocytes present in the focal lesions (38). Tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and nitric oxide (NO) derivatives appear to be required for the suppression of salmonella growth in the RES (27, 28, 32, 36, 37, 48). TNF-α is needed for the recruitment of mononuclear cells in the tissues and for granuloma formation (32); IFN-γ can activate macrophages to kill salmonellae in vitro (20).The establishment of the plateau phase coincides with the development of hypersusceptibility to the toxic and lethal effects of bacterial lipopolysaccharide (LPS) (29, 33). We have previously shown that mice immunized with a live attenuated aromatic-dependent Salmonella vaccine strain show transient hypersusceptibility to LPS, which can be prevented by treatment with anti-TNF-α antibodies (29). The role of other cytokines in this phenomenon is not known.Interleukin-12 (IL-12) is a 70-kDa heterodimeric cytokine produced by macrophages, B cells, polymorphonuclear leukocytes, and dendritic cells in response to a variety of stimuli including products of bacterial origin (5, 10). IL-12 mediates resistance to intracellular organisms including Listeria, Toxoplasma, Candida, Leishmania, Mycobacterium tuberculosis, and Brucella abortus (8, 13, 18, 23, 39, 46, 50). IL-12 is generally believed to mediate host resistance by inducing IFN-γ production by NK and T cells as well as by contributing to the establishment of protective Th1 antigen-specific responses (5, 6, 9, 10, 12, 13, 24, 34, 39, 43, 47).Evidence for IL-12 induction in salmonellosis has been provided. IL-12 and IL-12-specific mRNA have been detected in vivo and in vitro in response to Salmonella. Elicited peritoneal mouse macrophages stimulated with Salmonella dublin express elevated levels of IL-12 p40-specific mRNA (4, 7). Oral infection with virulent or live attenuated S. dublin induces early (6 and 20 h postinfection) production of IL-12-specific mRNA in Peyer’s patches and mesenteric lymph nodes (3); biologically active IL-12 in lymph node homogenates has been documented 36 h after S. dublin infection (21). We and others previously reported that in vivo IL-12 neutralization reduces the ability of the host to suppress the growth of virulent salmonellae in the tissues and impairs IFN-γ production (21, 31). A recent report indicates that a mutation in the IL-12 receptors renders humans more susceptible to salmonellosis (11). Nevertheless, the mechanisms by which IL-12 mediates host resistance to Salmonella are still unclear.In the present study, we attempted to clarify the mechanisms by which IL-12 contributes to host resistance in mice infected with Salmonella. We investigated the role of IL-12 in survival, granuloma formation, and macrophage activation in mice infected with an attenuated Salmonella strain that normally causes very mild infections in BALB/c mice. We also investigated the involvement of IL-12 in the toxic and lethal effects of high bacterial loads in the tissues as well as in the expression of hypersusceptibility to LPS normally seen in mice infected with salmonellae. We also wished to clarify the involvement of IFN-γ in IL-12-mediated resistance to salmonellosis.     

ICAM-1 Is Necessary for Epithelial Recruitment of γδ T Cells and Efficient Corneal Wound Healing

Wound healing and inflammation are both significantly reduced in mice that lack γδ T cells. Here, the role of epithelial intercellular adhesion molecule-1 (ICAM-1) in γδ T cell migration in corneal wound healing was assessed. Wild-type mice had an approximate fivefold increase in epithelial γδ T cells at 24 hours after epithelial abrasion. ICAM-1^−/− mice had 50.9% (P < 0.01) fewer γδ T cells resident in unwounded corneal epithelium, which failed to increase in response to epithelial abrasion. Anti-ICAM-1 blocking antibody in wild-type mice reduced epithelial γδ T cells to a number comparable to that of ICAM-1^−/− mice, and mice deficient in lymphocyte function-associated antigen-1 (CD11a/CD18), a principal leukocyte receptor for ICAM-1, exhibited a 48% reduction (P < 0.01) in peak epithelial γδ T cells. Re-epithelialization and epithelial cell division were both significantly reduced (∼50% at 18 hours, P < 0.01) after abrasion in ICAM-1^−/− mice versus wild-type, and at 96 hours, recovery of epithelial thickness was only 66% (P < 0.01) of wild-type. ICAM-1 expression by corneal epithelium in response to epithelial abrasion appears to be critical for accumulation of γδ T cells in the epithelium, and deficiency of ICAM-1 significantly delays wound healing. Since γδ T cells are necessary for efficient epithelial wound healing, ICAM-1 may contribute to wound healing by facilitating γδ T cell migration into the corneal epithelium.Intercellular adhesion molecule-1 (ICAM-1, CD54)¹ is a conserved member of the immunoglobulin supergene family² and is expressed by many cell types in response to stimuli such as cytokines,^3,4 and oxidative and physical stress.^5,6 It has been extensively studied in the context of adhesion and transmigration of leukocytes through endothelium⁷ and epithelium,^8,9 and it also serves as an adhesive ligand for leukocyte-mediated cytotoxic activity.^9,10,11 ICAM-1 is recognized by members of the β2 (CD18) integrin family, especially lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18),¹² and this adhesion is critical to many of the migratory and cytotoxic events in which ICAM-1 participates.^7,10,11 ICAM-1 also functions as a signaling molecule, dependent on its cytoplasmic tail interacting with cytoskeletal elements.⁷ This capability influences functions such as leukocyte transendothelial migration⁷ and vascular permeability.¹³Of importance to the current study is the fact that ICAM-1 can be expressed by corneal epithelial cells and limbal vessel endothelial cells.^{14,15,16,17,18,19,20,21} It appears to be expressed in conditions associated with inflammation, but its role in this context is poorly understood, especially its expression by the epithelial cells. Using a murine model of central corneal epithelial abrasion, we observed ICAM-1 on corneal epithelial cells in the periphery of the cornea, a region not directly injured by the abrasion.¹⁴ Since migration and division of these cells account for wound closure and re-establishment of full thickness epithelium necessary for healing,^22,23 it was of interest to determine whether ICAM-1 is necessary for these processes. To this end we studied wound healing in mice that do not express ICAM-1.^24,25As a part of this evaluation, we focused attention on γδ T cells. We observed in earlier studies that epithelial expression of ICAM-1 occurred at a time when γδ T cells increased within the corneal epithelium,^14,26 and that γδ T cell-deficient mice exhibited poor corneal wound healing. Since these leukocytes express LFA-1,²⁷ and LFA-1/ICAM-1 interactions support adhesion of human lymphocytes to human epithelial cells expressing ICAM-1,^20,27 it seemed possible that γδ T cell accumulation in the epithelium after corneal abrasion would be influenced by the absence of ICAM-1.     

Characteristics of Invasive Candidiasis in Gamma Interferon- and Interleukin-4-Deficient Mice: Role of Macrophages in Host Defense against Candida albicans

Murine models of invasive candidiasis were used to study the in vivo importance of gamma interferon (IFN-γ) and interleukin-4 (IL-4) in host defense against Candida albicans and to characterize the tissue inflammatory reactions, with special reference to macrophages (Mφ). Knockout (KO) IFN-γ-deficient (GKO) and IL-4-deficient (IL-4 KO) and C57BL/6 parental mouse strains were challenged intraperitoneally with 10⁸ C. albicans blastoconidia. Survival of GKO mice was significantly lower (16.7%) than that of C57BL/6 control (55.5%) and IL-4 KO (61.1%) animals, but was not correlated with the extent of organ colonization. Immunohistological analysis with a panel of myeloid and lymphoid markers revealed multiple renal abscesses, myocarditis, hepatitis, meningoencephalitis, and pneumonia in each strain, with a dominant presence of Mφ. In the absence of IFN-γ, C. albicans induced striking changes in the phenotype of alveolar Mφ and extensive perivascular lymphoid infiltrates in the lung. Impairment in nitric oxide production by peritoneal Mφ was shown only in GKO mice, and they produced Candida-specific immunoglobulin G (IgG), IgM, IgA, and IgG subclasses in lower titers. Our in vivo studies with KO mice elucidate a critical role for IFN-γ, but not IL-4, in host defense against C. albicans.Candida albicans is a common commensal organism in humans, and its importance as an opportunistic pathogen, particularly in immunocompromised patients, has continued to increase over the last two decades. According to the National Nosocomial Infections Surveillance System, the ratio of C. albicans isolates among nosocomial fungal infections increased from 52% to 63% in the 1980s (4). Phagocytic cell defects generally predispose to disseminated candidiasis; candidemia was calculated to result in 38% excess mortality and extend hospitalization by approximately 30 days (40). Besides the efforts to develop more effective and safer antifungal agents, a new therapeutic approach to augment the antifungal capacity of the host’s immune system should be investigated.The mechanisms of host defense and pathogenesis of candidiasis are not completely understood. Optimal phagocytosis of C. albicans requires opsonization; however, unopsonized yeast can be internalized by macrophages (Mφ) through the mannose receptor (21). Efficient killing of C. albicans by mononuclear phagocytes requires respiratory burst-associated toxic compounds (22), and recent data suggest that nitric oxide (NO) may also be involved in anticandidal functions of Mφ (5). Experimental evidence suggests that mononuclear phagocytes could play an important role in eradication of this pathogen, and their anticandidal activity can be augmented in vitro with granulocyte-Mφ and Mφ colony-stimulating factors and cytokines (no significant change could be measured in the level of specific immunoglobulin A [IgA] in serum or among the levels of interleukin-3 [IL-3] and gamma interferon [IFN-γ]) in both human and murine systems (23, 25, 28, 39).The in vivo benefit of cytokine treatment in disseminated candidiasis has not been established, and data from different murine models are controversial. Administration of IFN-γ has been reported to be associated with improved survival of mice after lethal challenge with C. albicans, which correlated with the anticandidal activity of peritoneal Mφ (28); another study showed a reduction in tissue fungal burden in IFN-γ-treated mice (19). However, in a different murine model, in vivo administration of IFN-γ resulted in increased susceptibility and organ colonization of four infected inbred strains (13). In vivo administration of IL-12, which has been reported to prime naive T cells for high IFN-γ expression and skew cytokine production toward a Th1-type response (38), did not modify the course of systemic candidiasis (32). In contrast, Th2-type cytokines IL-4 and IL-10 have been reported to exacerbate infection, and neutralization of IL-4 by specific antibody or soluble IL-4 receptor resulted in an enhanced production of Th1 cytokines, associated with increased resistance to systemic murine candidiasis (26, 30, 37). The controversial results of in vivo cytokine treatment may be the consequence of genetic differences among the infected strains and also the variation in protocols; the kinetics of cytokine production are influenced by several host and pathogen factors, and the effect of exogenous cytokine might depend on the condition of the infected host and stage of infection.Cytokine and receptor gene disruption strategies make it possible to examine the role of cytokines in host response to different pathogens directly. Recent studies showed an increased susceptibility of IFN-γ–receptor knockout (KO) mice to Mycobacterium bovis or Mycobacterium tuberculosis, but not to Schistosoma mansoni (1, 7, 8). Another study reported that disruption of the IFN-γ receptor gene was associated with higher susceptibility to Leishmania major and that IL-4 deficiency resulted in increased resistance, but only in certain inbred strains (17).Our study was undertaken to investigate the in vivo role of IFN-γ and IL-4 in disseminated C. albicans infection and characterize the tissue inflammatory cells by immunohistochemistry and by functional assays ex vivo. We demonstrate that IFN-γ, but not IL-4, is essential for survival in invasive candidiasis and show the dominant participation of Mφ in the inflammatory lesions of different tissues in KO as well as wild-type mice. In the absence of IFN-γ, a striking local immune regulatory alteration was observed in the lungs.     

Suppressed Accumulation of Cerebral Amyloid β Peptides in Aged Transgenic Alzheimer’s Disease Mice by Transplantation with Wild-Type or Prostaglandin E2 Receptor Subtype 2-Null Bone Marrow

A complex therapeutic challenge for Alzheimer’s disease (AD) is minimizing deleterious aspects of microglial activation while maximizing beneficial actions, including phagocytosis/clearance of amyloid β (Aβ) peptides. One potential target is selective suppression of microglial prostaglandin E₂ receptor subtype 2 (EP2) function, which influences microglial phagocytosis and elaboration of neurotoxic cytokines. To test this hypothesis, we transplanted bone marrow cells derived from wild-type mice or mice homozygous deficient for EP2 (EP2^−/−) into lethally irradiated 5-month-old wild-type or APPswe-PS1ΔE9 double transgenic AD mouse model recipients. We found that cerebral engraftment by bone marrow transplant (BMT)-derived wild-type or EP2^−/− microglia was more efficient in APPswe-PS1ΔE9 than in wild-type mice, and APPswe-PS1ΔE9 mice that received EP2^−/− BMT had increased cortical microglia compared with APPswe-PS1ΔE9 mice that received wild-type BMT. We found that myeloablative irradiation followed by bone marrow transplant-derived microglia engraftment, rather than cranial irradiation or BMT alone, was responsible for the approximate one-third reduction in both Aβ plaques and potentially more neurotoxic soluble Aβ species. An additional 25% reduction in cerebral cortical Aβ burden was achieved in mice that received EP2^−/− BMT compared with mice that received wild-type BMT. Our results provide a foundation for an adult stem cell-based therapy to suppress soluble Aβ peptide and plaque accumulation in the cerebrum of patients with AD.Alzheimer’s disease (AD), the most common dementing neurodegenerative disease,¹ is a major public health burden for older Americans.² Amyloid β (Aβ) peptides are pleotropic molecules that are directly neurotoxic and stimulate liberation of cytotoxic cytokines through activation of microglia innate immune response.³ However, activated microglia phagocytosis and degradation of Aβ species is key to cerebral Aβ homeostasis.⁴ Thus, an important but complex therapeutic challenge is balancing deleterious and beneficial aspects of microglial activation in AD.⁵ One proposed mechanism of microglial modulation is prostaglandin E₂ signaling, especially through activation of the E prostanoid receptor subtype 2 (EP2).⁶ Cultured microglia lacking EP2 (EP2^−/−) show enhanced phagocytosis of Aβ from human brain explants and reduced paracrine neurotoxicity.⁷ In vivo experiments with EP2^−/− mice have shown reduced accumulation of cerebral Aβ in a transgenic mouse model of AD,^7,8,9 as well as suppressed oxidative damage to neurons following innate immune activation.^7,10,11,12 However, because EP2 is expressed by several cell types in brain, including microglia and neurons, the importance of microglial-specific EP2 has not been established. To address this gap in our knowledge, bone marrow cells from EP2^−/− mice were transplanted into APPswe-PS1ΔE9 mice.Circulating bone marrow transplant (BMT)-derived cells can selectively replace resident microglia,¹³ and up to 30% of microglia can be derived from donor marrow in wild-type mice recipients up to a year after transplantation.^14,15 Moreover, engraftment of brain appears qualitatively more efficient in recipient AD mice than in wild-type controls.^16,17 The reasons for the apparent higher engraftment are not clear, but may be in response to chronic low level immune activation in AD mouse brains.^16,17 Some investigators have shown BMT-derived microglia associated with Aβ deposits in vivo, and that transgenic AD mouse BMT recipients have reduced Aβ plaque burden.¹⁷ Although previous data addressed potential mechanisms by which BMT-derived microglia might promote clearance of Aβ peptides,¹⁸ the results of these studies were confounded by the effects of preconditioning brain irradiation; it is possible that the reduced Aβ plaque burden was caused by irradiation-induced alteration of Aβ production or clearance rather than BMT-derived microglia. In the current studies, we robustly quantify microglial engraftment in brains of APPswe-PS1ΔE9 mice. In addition, we control for the potential confounder of irradiation-mediated Aβ peptide suppression by evaluating Aβ in mice that received cranial-specific irradiation with or without BMT. Finally, we test the hypothesis that BMT with cells from EP2^−/− mice would enhance cerebral bone marrow derived microglia engraftment and clearance of Aβ peptides from cerebrum of APPswe-PS1ΔE9 mice.     

Aggregation and Binding Substances Enhance Pathogenicity in Rabbit Models of Enterococcus faecalis Endocarditis

We investigated the importance of enterococcal aggregation substance (AS) and enterococcal binding substance (EBS) in rabbit models of Enterococcus faecalis cardiac infections. First, American Dutch belted rabbits were injected intraventricularly with 10⁸ CFU and observed for 2 days. No clinical signs of illness developed in animals given AS⁻ EBS⁻ organisms, and all survived. All rabbits given AS⁻ EBS⁺ organisms developed signs of illness, including significant pericardial inflammation, but only one of six died. All animals given AS⁺ EBS⁻ organisms developed signs of illness, including pericardial inflammation, and survived. All rabbits given AS⁺ EBS⁺ organisms developed signs of illness and died. None of the rabbits receiving AS⁺ EBS⁺ organisms showed gross pericardial inflammation. The lethality and lack of inflammation are consistent with the presence of a superantigen. Rabbit and human lymphocytes were highly stimulated in vitro by cell extracts, but not cell-free culture fluids, of AS⁺ EBS⁺ organisms. In contrast, cell extracts from AS⁻ EBS⁻ organisms weakly stimulated lymphocyte proliferation. Culture fluids from human lymphocytes stimulated with AS⁺/EBS⁺ enterococci contained high levels of gamma interferon and tumor necrosis factor alpha (TNF-α) and TNF-β, which is consistent with functional stimulation of T-lymphocyte proliferation and macrophage activation. Subsequent experiments examined the abilities of the same strains to cause endocarditis in a catheterization model. New Zealand White rabbits underwent transaortic catheterization for 2 h, at which time catheters were removed and animals were injected with 2 × 10⁹ CFU of test organisms. None of the animals given AS⁻ EBS⁻ organisms developed vegetations or showed autopsy evidence of tissue damage. Rabbits given AS⁻ EBS⁺ or AS⁺ EBS⁻ organisms developed small vegetations and had splenomegaly at autopsy. All rabbits given AS⁺ EBS⁺ organisms developed large vegetations and had splenomegaly and lung congestion at autopsy. Similar experiments that left catheters in place for 3 days revealed that all rabbits given AS⁻ EBS⁻ or AS⁺ EBS⁺ organisms developed vegetations, but animals given AS⁺ EBS⁺ organisms had larger vegetations and autopsy evidence of lung congestion. These experiments provide direct evidence that these two cell wall components play an important role in the pathogenesis of endocarditis as well as in conjugative plasmid transfer.Recently, Enterococcus faecalis and other enterococci have become increasingly recognized as significant causes of nosocomial infections (23, 26, 27, 30). They are important causes of bacteremia, endocarditis, and urinary tract infections. These organisms are also important because of their increasing incidence of resistance to vancomycin and other antibiotics and because of the potential of transferring antibiotic resistance to other bacteria.An important mechanism for horizontal transfer of antibiotic resistance in enterococci is pheromone-inducible conjugation (10, 12, 34). The expression of conjugative transfer functions of plasmids such as pCF10 (58 kb; encodes tetracycline resistance [12, 14]) and pAD1 (60 kb; encodes hemolysin and bacteriocin production [10, 31]) is induced by peptide pheromones produced by recipient cells (13, 31, 34). The conjugation gene products induced by pheromones include a cell surface adhesin, aggregation substance (AS). This protein mediates the formation of mating aggregates between donor and recipient cells by binding to a cognate ligand on the recipient cell, enterococcal binding substance (EBS) (13, 14). The prgB gene of pCF10 encodes the AS protein, Asc10 (25), whose nucleotide and amino acid sequences are highly similar to those of AS proteins encoded by other pheromone plasmids (19, 20). The genetics of EBS are complex, with multiple, unlinked insertion mutations required to generate an EBS-negative phenotype (5, 12, 32). Lipoteichoic acid (LTA) appears to be an important component of EBS (7, 15, 32).Previous studies of the pathogenicity of E. faecalis have shown that hemolysin contributes to the virulence of the organism in animal models, including murine peritonitis, rabbit endophthalmitis, and rabbit endocarditis (9, 11, 21, 25, 26). In their study, Chow et al. (9) also showed that AS contributed significantly to the production of experimental endocarditis. Hemolysin and AS were associated with increased mortality, and AS was associated with increased vegetation weight.AS proteins of E. faecalis are thought to be virulence factors in enterococcal infections by promoting binding to a variety of eukaryotic cell surfaces (21, 25, 26). AS expression may be induced in vivo by eukaryotic factors in serum (6). AS contains amino acid motifs, Arg-Gly-Asp-Ser and Arg-Gly-Asp-Val, which are found in fibronectin and other proteins and which mediate binding to eukaryotic cell adhesion molecules of the integrin superfamily (18, 21, 25). Soluble LTA inhibits aggregate formation and may function as EBS (26). LTA from E. faecalis has previously been shown to induce both interleukin 1β and tumor necrosis factor alpha (TNF-α) production from macrophages (7).This study was undertaken to evaluate the role of both AS and EBS in two rabbit models of E. faecalis cardiac infections. Greater insight into the role of these two factors in virulence may lead to alternative methods of prophylaxis and treatment of resistant enterococcal infections. Our studies indicate that the presence of both cell surface components is associated with both increased mortality and formation of vegetations.(This work was presented in part at the 13th Lancefield International Symposium on Streptococci and Streptococcal Diseases, Paris, France, 16 to 20 September 1996.)     

Roles for NK Cells and an NK Cell-Independent Source of Intestinal Gamma Interferon for Innate Immunity to Cryptosporidium parvum Infection

A gamma interferon (IFN-γ)-dependent innate immune response operates against the intestinal parasite Cryptosporidium parvum in T- and B-cell-deficient SCID mice. Although NK cells are a major source of IFN-γ in innate immunity, their protective role against C. parvum has been unclear. The role of NK cells in innate immunity was investigated using Rag2^−/− mice, which lack T and B cells, and Rag2^−/− γ_c^−/− mice, which, in addition, lack NK cells. Adult mice of both knockout lines developed progressive chronic infections; however, on most days the level of oocyst excretion was higher in Rag2^−/− γ_c^−/− mice and these animals developed morbidity and died, whereas within the same period the Rag2^−/− mice appeared healthy. Neonatal mice of both mouse lines survived a rapid onset of infection that reached a higher intensity in Rag2^−/− γ_c^−/− mice. Significantly, similar levels of intestinal IFN-γ mRNA were expressed in Rag2^−/− and Rag2^−/− γ_c^−/− mice. Also, infections in each mouse line were exacerbated by treatment with anti-IFN-γ neutralizing antibodies. These results support a protective role for NK cells and IFN-γ in innate immunity against C. parvum. In addition, the study implies that an intestinal cell type other than NK cells may be an important source of IFN-γ during infection and that NK cells may have an IFN-γ-independent protective role.Cryptosporidiosis is an infectious diarrheal disease that affects different types of vertebrates, including mammals (3). The etiological agent is the monoxenous protozoan parasite Cryptosporidium, which belongs to the Apicomplexa. One species, Cryptosporidium hominis, may have a predilection for infecting humans, while a morphologically similar parasite, Cryptosporidium parvum, readily infects both cattle and humans (3). The cryptosporidia of mammals invade intestinal epithelial cells, where they multiply asexually to produce merozoites that infect more cells. Eventually, merozoites may undergo differentiation into gamonts that form new oocysts, containing four sporozoites, and the oocysts transmit infection to new hosts by the fecal-oral route. The clinical phase of cryptosporidiosis normally lasts a few days but may persist and become fatal in immunocompromised hosts (2).Studies of protective host immune responses to Cryptosporidium indicate that elimination of infection involves adaptive immunity and, in particular, requires the presence of CD4⁺ T cells. AIDS patients with low CD4⁺ cell counts have shown increased susceptibility to cryptosporidial infection and high rates of morbidity and mortality, while resolution of AIDS-associated infection following anti-human-immunodeficiency-virus drug treatment coincided with the partial recovery of intestinal CD4⁺ T-cell counts (2, 23). Mice with a CD4⁺ T-cell deficiency were found to be incapable of clearing C. parvum infection (1), and similarly, depletion of these cells from immunocompetent animals with specific antibody increased oocyst production (27). CD4⁺ T cells are also an important source of gamma interferon (IFN-γ), and this cytokine plays a key role in the control of infection. Antigen-specific IFN-γ production by restimulated CD4⁺ T cells from humans who recovered from infection was observed, although cells taken during acute infection were not responsive to antigen (6). IFN-γ^−/− mice or mice administered anti-IFN-γ neutralizing antibodies had exacerbated infections compared with control animals (18, 27). IFN-γ activity during C. parvum infection has been associated with a chemokine response by intestinal epithelial cells that attracted both CD4⁺ T cells and macrophages into the lamina propria (10). In addition, IFN-γ has been shown to have a direct effect on parasite growth by activating epithelial cell antimicrobial killing activity (19).Innate immune responses are also able to limit the reproduction of C. parvum. Immunocompromised adult nude mice (lacking T cells) or SCID mice (lacking T and B cells) developed chronic infections that were controlled for a number of weeks but eventually became progressive and fatal (13, 17, 27). IFN-γ was important for the initial resistance of these mice, since administration of anti-IFN-γ neutralizing antibodies to adult or neonatal SCID mice increased susceptibility to infection (14, 28), and repeated antibody treatment resulted in rapid establishment of severe infection (14). In addition, morbidity as a result of parasite reproduction appeared sooner in SCID IFN-γ^−/− mice than in SCID mice (7).NK cells are involved in resistance to intracellular microbial pathogens, including protozoa, and are a major source of IFN-γ in innate immunity (9). NK cells originate mainly in the bone marrow, from where they migrate to other organs (5, 29). Interleukin-15 (IL-15) is essential for differentiation and subsequent survival of NK cells and can also be important in activation of the cells (5, 9). NK cells are activated by ancillary cells, such as dendritic cells (DCs), by direct contact and by proinflammatory cytokines produced by DCs stimulated by antigen (9). Activated NK cells produce IFN-γ and other proinflammatory cytokines and may also become cytotoxic against infected cells.The protective role of NK cells in innate immunity to C. parvum is unclear, but some studies imply that these cells may be involved. Human peripheral blood NK cells treated with IL-15 were shown to have cytolytic activity against human intestinal epithelial cell lines infected with C. parvum (4), and intestinal expression of this cytokine has been detected in humans (20). C. parvum infection was found to be more widespread in SCID mice deficient in NK cell cytotoxicity than in SCID mice with normal NK cell function (17). In addition, in vitro studies demonstrated that splenocytes from SCID mice produced IFN-γ in the presence of cryptosporidial antigens, but if NK cells were depleted, IFN-γ production did not occur (15). However, attempts to show that NK cells were protective in SCID mice infected with C. parvum have not been successful. In separate studies, treatment of these mice with anti-asialo-GM1 antibodies that can deplete NK cells in vivo was shown to have no effect on the course of C. parvum infection (15, 27), and while it has been argued that these antibodies might not have reached the gut in sufficient quantity to be effective, similar antibodies were shown to diminish intestinal NK cell function (30).The aim of the present study was to examine further the role of NK cells and IFN-γ in the innate immune response to C. parvum. The pattern of infection and immune responses were compared in Rag2^−/− mice, which lack T and B cells, and Rag2^−/− γ_c^−/− mice, which, in addition, lack NK cells due to the absence of the γ_c chain component of the IL-15 receptor (5). The results support protective roles for IFN-γ and NK cells in innate immunity to C. parvum but also indicate that IFN-γ from a cell type other than NK cells is important for control of infection.     

IL-4/IL-13-Dependent Alternative Activation of Macrophages but Not Microglial Cells Is Associated with Uncontrolled Cerebral Cryptococcosis

Both interleukin (IL)-4- and IL-13-dependent Th2-mediated immune mechanisms exacerbate murine Cryptococcus neoformans-induced bronchopulmonary disease. To study the roles of IL-4 and IL-13 in cerebral cryptococcosis, IL-4 receptor α-deficient (IL-4Rα^−/−), IL-4-deficient (IL-4^−/−), IL-13-deficient (IL-13^−/−), IL-13 transgenic (IL-13^T/+), and wild-type mice were infected intranasally. IL-13^T/+ mice displayed a higher fungal brain burden than wild-type mice, whereas the brain burdens of IL-4Rα^−/−, IL-4^−/−, and IL-13^−/− mice were significantly lower as compared with wild-type mice. On infection, 68% of wild-type mice and 88% of IL-13-overexpressing IL-13^T/+ mice developed significant cerebral lesions. In contrast, only a few IL-4Rα^−/−, IL-4^−/−, and IL-13^−/− mice had small lesions in their brains. Furthermore, IL-13^T/+ mice harbored large pseudocystic lesions in the central nervous system parenchyma, bordered by voluminous foamy alternatively activated macrophages (aaMphs) that contained intracellular cryptococci, without significant microglial activation. In wild-type mice, aaMphs tightly bordered pseudocystic lesions as well, and these mice, in addition, showed microglial cell activation. Interestingly, in resistant IL-4^−/−, IL-13^−/−, and IL-4Rα^−/− mice, no aaMphs were discernible. Microglial cells of all mouse genotypes neither internalized cryptococci nor expressed markers of alternative activation, although they displayed similar IL-4Rα expression levels as macrophages. These data provide the first evidence of the development of aaMphs in a central nervous system infectious disease model, pointing to distinct roles of macrophages versus microglial cells in the central nervous system immune response against C. neoformans.The opportunistic pathogenic yeast Cryptococcus neoformans causes life-threatening fungal infections of most internal organs including the central nervous system (CNS), primarily in patients affected by immunodeficiency syndromes such as AIDS.¹ The pathogenesis of cryptococcosis is not fully understood, however, especially in cases of different levels of immunocompetence. It is generally accepted that the fungus first invades the respiratory system, where it leads to relatively mild or asymptomatic bronchopneumonia in the immunocompetent.^2,3,4,5 Fungemia with generalization of the infection may result from reduced immunological control mechanisms.^6,7,8,9 Invasion of the CNS with subsequent development of meningoencephalitis is the major cause of death during cryptococcosis.^10,11The precise reaction pattern of recruited inflammatory cells, especially monocytes/macrophages, due to fungal invasion of the CNS parenchyma has been addressed mainly via analysis of helper T cell (Th)1 responses.¹² In this context, in addition to protective Th1-driven immune responses, the role of Th2 cytokines has gained interest recently.¹³ The major Th2 cytokines interleukin (IL)-4 and IL-13 act via the IL-4Rα chain together with the γ_c chain or the IL-13Rα1/2 chains, and regulate macrophage functional status.¹⁴ IL-4 has been shown to be detrimental in murine models of systemic and pulmonary cryptococcosis,^{6,15,16,17,18} and we have recently illustrated the role of IL-13 in inducing the formation of alternatively activated macrophages (aaMphs) in murine pulmonary cryptococcosis.¹⁹The activation phenotype of macrophages may critically influence the regulatory mechanisms by which inflammation and infection in the CNS are controlled. According to the current paradigm, classically activated macrophages are primed by interferon-γ and produce tumor necrosis factor, IL-1, oxygen and nitrogen radicals,²⁰ thereby producing proinflammatory cytokines that regulate the Th1 immune response. In contrast, aaMph²¹ develop in response to Th2 cytokine stimulation such as IL-4 and IL-13 and are characterized by expression of genes associated with endocytosis and tissue repair such as arginase-1, mannose receptor (CD206), found-in-inflammatory-zone (FIZZ), and chitinase 3-like 3 (YM1) and largely fail to produce nitric oxide (NO) due to their induction of arginase.²² As such, they are thought to be involved in tissue repair and remodeling,^22,23 in protection against diet-induced obesity,^24,25 and schistosomiasis,²⁶ but they may also elicit adverse tissue processes such as pulmonary or liver fibrosis.^{27,28,29,30,31} In particular, their development renders the host vulnerable to infection with pathogens where macrophage activation and killing functions are required.³²In murine models of pulmonary C. neoformans infection, aaMph have been shown to be associated with uncontrolled lung infection.^18,19 The role of aaMph versus classically activated macrophage in the CNS due to pulmonary infection with the neurotropic pathogen C. neoformans has not been defined yet. In this study, we aimed to characterize the morphology and functional status of CNS macrophages in cerebral cryptococcosis following intranasal infection of susceptible wild-type and IL-13-transgenic BALB/c mice. Moreover, using mice unable to produce IL-4 or IL-13 or respond to both (IL-4Rα^−/− mice), we show that abrogation of CNS aaMph development is associated with controlled infection.     

Processing of Mycobacterium tuberculosis Bacilli by Human Monocytes for CD4+ αβ and γδ T Cells: Role of Particulate Antigen

Mycobacterium tuberculosis readily activates both CD4⁺ and Vδ2⁺ γδ T cells. Despite similarity in function, these T-cell subsets differ in the antigens they recognize and the manners in which these antigens are presented by M. tuberculosis-infected monocytes. We investigated mechanisms of antigen processing of M. tuberculosis antigens to human CD4 and γδ T cells by monocytes. Initial uptake of M. tuberculosis bacilli and subsequent processing were required for efficient presentation not only to CD4 T cells but also to Vδ2⁺ γδ T cells. For γδ T cells, recognition of M. tuberculosis-infected monocytes was dependent on Vδ2⁺ T-cell-receptor expression. Recognition of M. tuberculosis antigens by CD4⁺ T cells was restricted by the class II major histocompatibility complex molecule HLA-DR. Processing of M. tuberculosis bacilli for Vδ2⁺ γδ T cells was inhibitable by Brefeldin A, whereas processing of soluble mycobacterial antigens for γδ T cells was not sensitive to Brefeldin A. Processing of M. tuberculosis bacilli for CD4⁺ T cells was unaffected by Brefeldin A. Lysosomotropic agents such as chloroquine and ammonium chloride did not affect the processing of M. tuberculosis bacilli for CD4⁺ and γδ T cells. In contrast, both inhibitors blocked processing of soluble mycobacterial antigens for CD4⁺ T cells. Chloroquine and ammonium chloride insensitivity of processing of M. tuberculosis bacilli was not dependent on the viability of the bacteria, since processing of both formaldehyde-fixed dead bacteria and mycobacterial antigens covalently coupled to latex beads was chloroquine insensitive. Thus, the manner in which mycobacterial antigens were taken up by monocytes (particulate versus soluble) influenced the antigen processing pathway for CD4⁺ and γδ T cells.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is spread readily from person to person by inhalation of aerosolized mycobacteria (8). A hallmark of M. tuberculosis infection is the ability of most healthy individuals to control the infection by mounting an acquired immune response, in which antigen-specific T cells and mononuclear phagocytes arrest the growth of M. tuberculosis bacilli and maintain control over dormant bacilli within granulomas (reviewed in reference 25). This protective cellular immune response results in conversion of the tuberculin skin test from negative to positive and probably in increased resistance to reinfection with tubercle bacilli.CD4⁺ αβ-T-cell-receptor (αβ TCR)-bearing T cells (CD4⁺ T cells) are readily activated by mycobacterial antigens and have a dominant role in the protective immune response to M. tuberculosis in humans (2, 34). These CD4⁺ T cells not only secrete cytokines but also serve directly as cytotoxic effector cells against M. tuberculosis-infected macrophages (6). In addition to CD4⁺ T cells, M. tuberculosis antigens activate other human T-cell subsets such as γδ TCR⁺ T cells (γδ T cells) (15, 16, 18). Vδ2⁺ and Vγ9⁺ γδ T cells are particularly responsive to live M. tuberculosis (15). A role for both γδ and CD4⁺ T cells in protective immunity to acute M. tuberculosis infection has been demonstrated in murine models (20, 21, 26, 27). A recent study of humans suggests that Vγ9⁺ and Vδ2⁺ γδ T-cell numbers and function are reduced in tuberculosis patients (23).Functional comparisons of human CD4⁺ and γδ T-cell responses of healthy tuberculin-positive persons demonstrate that both T-cell subsets have similar cytotoxic effector functions for M. tuberculosis-infected monocytes and produce large amounts of gamma interferon (IFN-γ), with γδ T cells being slightly more efficient producers of IFN-γ than CD4⁺ T cells (37). Despite similarities in function, these two T-cell subsets differ in the mycobacterial antigens recognized by their TCRs and the manners in which antigens are presented to them by M. tuberculosis-infected mononuclear phagocytes. CD4⁺ T cells recognize a wide diversity of mycobacterial peptides in the context of class II major histocompatibility complex (MHC) molecules, which include secreted as well as somatic antigens (6, 13, 33, 37). In contrast, Vγ9⁺ and Vδ2⁺ γδ T cells, the dominant γδ TCR subsets activated by M. tuberculosis, recognize mycobacterial antigens in a non-MHC-restricted manner and the repertoire of antigens includes small phosphate-containing antigens such as TUBag’s (5, 9, 19, 22, 29, 36).Both blood monocytes and alveolar macrophages infected with M. tuberculosis are efficient antigen-presenting cells for mycobacterial antigen-specific CD4⁺ and γδ T cells (1, 5). However, little is known about how M. tuberculosis-infected mononuclear phagocytes process antigens for these two T-cell subsets. M. tuberculosis bacilli are taken up by mononuclear phagocytes through a variety of surface receptors, including complement receptor 4, mannose receptor, and complement receptor 3 (17, 31, 32). Within mononuclear phagocytes, the mycobacteria reside within phagosomes and modulate the phagosome by preventing fusion with acidic lysosomal compartments (7). Although the vacuolar membranes surrounding the phagosome acquire endosomal markers, the vesicular proton ATPase is actively excluded, resulting in an elevated pH of 6.3 to 6.5 compared to the normal lysosomal pH of 4.5 (7, 35). The elevated pH in the phagosome does not appear to inhibit the ability of mycobacterial antigens to be processed and presented to CD4⁺ and Vδ2⁺ γδ T cells. This study was undertaken to gain insight into the mechanisms used by monocytes infected with live M. tuberculosis bacilli to process mycobacterial antigens for presentation to both CD4⁺ and γδ T cells.     

Reduction of IKKα Expression Promotes Chronic Ultraviolet B Exposure-Induced Skin Inflammation and Carcinogenesis

Ultraviolet B light (UVB) is a common cause of human skin cancer. UVB irradiation induces mutations in the tumor suppressor p53 gene as well as chronic inflammation, which are both essential for UVB carcinogenesis. Inhibitor of nuclear factor κB kinase-α (IKKα) plays an important role in maintaining skin homeostasis, and expression of IKKα was found to be down-regulated in human and murine skin squamous cell carcinomas. However, the role of IKKα in UVB skin carcinogenesis has not been investigated. Thus, here we performed UVB carcinogenesis experiments on Ikkα^+/+ and Ikkα^+/− mice. Ikkα^+/− mice were found to develop a twofold greater number of skin tumors than Ikkα^+/+ mice after chronic UVB irradiation. In addition, tumor latency was significantly shorter and tumors were bigger in Ikkα^+/− than in Ikkα^+/+ mice. At an early stage of carcinogenesis, an increase in UVB-induced p53 mutations as well as macrophage recruitment and mitogenic activity, and a decrease in UVB-induced apoptosis, were detected in Ikkα^+/− compared with those in Ikkα^+/+ skin. Also, reduction of IKKα levels in keratinocytes up-regulated the expression of monocyte chemoattractant protein-1 (MCP-1/CCL2), TNFα, IL-1, and IL-6, and elevated macrophage migration, which might promote macrophage recruitment and inflammation. Therefore, these findings suggest that reduction of IKKα expression orchestrates UVB carcinogen, accelerating tumorigenesis.Ultraviolet B (UVB) irradiation induces DNA damage. The tumor suppressor gene p53 is an UVB target, and human cutaneous squamous cell carcinoma (SCC) cells contain p53 mutations.¹ Skin cells can harbor UVB-induced p53 mutations for decades before the onset of human SCC, however,^2,3 underscoring the importance of coactivators in skin tumorigenesis. In mice, p53 mutations are an early genetic event in UVB skin carcinogenesis, which recapitulates the process of human SCC development.^4,5 The p53 mutations have been proposed to be important for UVB carcinogenesis because prevention of these mutations prevents skin tumor development, and the number of p53 mutation-positive keratinocytes correlates with the number of skin tumors in mice.^6,7,8,9 UVB exposure also induces chronic inflammation, cell proliferation, oxidant stress, and immunosuppression, which essentially facilitate UVB carcinogenesis.^10,11 Particularly, chronic inflammation can create a microenvironment that is prone to cell proliferation and DNA damage, thereby promoting tumor development.¹²On the other hand, UVB exposure also elicits protective responses, such as cell cycle arrest, DNA repair, and apoptosis, which reduce UVB-induced damage.^7,10,11 Previously, it was reported that mice lacking p53 were defective in inducing apoptosis after UVB irradiation and thus had more skin tumors than wild-type mice did.^13,14 Mice lacking Fas ligand had defects in apoptosis, which increased numbers of cells containing UVB-induced p53 mutations, and the mutant mice were more susceptible to UVB-induced skin tumors than wild-type mice.^5,8 Therefore, the different defects in inducing protective responses against UVB-induced damage can amplify the severity of the cancer cause, thereby accelerating carcinogenesis.Inhibitor of nuclear factor κB kinase-α (IKKα) is required for embryonic skin development in mice.^15,16 Several laboratories have reported the down-regulated expression and altered localization of IKKα protein as well as deletions and mutations in the Ikkα gene in human SCCs of the skin, lung, esophagus, and head and neck.^{17,18,19,20,21} In particular, IKKα deletion in keratinocytes was found to elevate an autocrine loop activity of epidermal growth factor receptor (EGFR), Ras, extracellular signal-related kinase (ERK), EGFR ligands, and the ligands’ activators and induced spontaneous skin SCCs in mice.²² Reduction of IKKα expression was found to promote chemical carcinogen-induced skin tumorigenesis.²³ Also, IKKα is an UVB-induced gene, and the defect in IKKα function was linked to inflammation.^24,25 UVB is a very common cause of human skin cancer; however, the role of IKKα in skin UVB carcinogenesis is largely unknown. Thus, here we examined the effect of reduced IKKα expression on UVB skin carcinogenesis in Ikkα^+/− and Ikkα^+/+ mice. Ikkα^+/− mice were significantly susceptible to UVB skin carcinogenesis than were Ikkα^+/+ mice. Because the tumor latency was significantly shorter and many more tumors were found in Ikkα^+/− mice than in Ikkα^+/+ mice, we analyzed the early events during UVB skin carcinogenesis. This study provided the first evidence showing the importance of IKKα in UVB skin carcinogenesis.     

γδ T Cells but Not NK Cells Are Essential for Cell-Mediated Immunity against Plasmodium chabaudi Malaria

Blood-stage Plasmodium chabaudi infections are suppressed by antibody-mediated immunity and/or cell-mediated immunity (CMI). To determine the contributions of NK cells and γδ T cells to protective immunity, C57BL/6 (wild-type [WT]) mice and B-cell-deficient (J_H^−/−) mice were infected with P. chabaudi and depleted of NK cells or γδ T cells with monoclonal antibody. The time courses of parasitemia in NK-cell-depleted WT mice and J_H^−/− mice were similar to those of control mice, indicating that deficiencies in NK cells, NKT cells, or CD8⁺ T cells had little effect on parasitemia. In contrast, high levels of noncuring parasitemia occurred in J_H^−/− mice depleted of γδ T cells. Depletion of γδ T cells during chronic parasitemia in B-cell-deficient J_H^−/− mice resulted in an immediate and marked exacerbation of parasitemia, suggesting that γδ T cells have a direct killing effect in vivo on blood-stage parasites. Cytokine analyses revealed that levels of interleukin-10, gamma interferon (IFN-γ), and macrophage chemoattractant protein 1 (MCP-1) in the sera of γδ T-cell-depleted mice were significantly (P < 0.05) decreased compared to hamster immunoglobulin-injected controls, but these cytokine levels were similar in NK-cell-depleted mice and their controls. The time courses of parasitemia in CCR2^−/− and J_H^−/− × CCR2^−/− mice and in their controls were nearly identical, indicating that MCP-1 is not required for the control of parasitemia. Collectively, these data indicate that the suppression of acute P. chabaudi infection by CMI is γδ T cell dependent, is independent of NK cells, and may be attributed to the deficient IFN-γ response seen early in γδ T-cell-depleted mice.Malaria remains a leading cause of morbidity and mortality, annually killing about 2 million people worldwide (32, 33). Despite decades of research, malaria is a reemerging disease because of increasing drug resistance by malarial parasites and insecticide resistance by the mosquito vector. Most infected individuals do not succumb to malaria but develop clinical immunity where parasite replication is controlled to some degree by the immune system without eliciting clinical disease or sterile immunity (14, 38).Understanding the immunologic pathways leading to the control of blood-stage parasite replication is important for defining the mechanisms of disease pathogenesis and improving vaccines currently in development. The early events of the immune response depend upon activation of the innate immune system, which regulates the downstream adaptive immune response needed to control or cure (44). Natural killer (NK) and γδ T cells function early in the immune response to pathogens as components of the innate immune system. Both cell types have been proposed to play significant roles in the subsequent clearance of blood-stage malarial parasites by activating the adaptive immune system (35, 43, 44). The mechanism by which they accomplish this appears to be mediated via their secretion of gamma interferon (IFN-γ) induced by cytokines such as interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-α), and IL-6 produced by other components of the innate immune system, including macrophages and dendritic cells (17, 25, 26, 37, 49).Blood-stage malaria parasites are cleared by mature isotypes of antibodies and/or by antibody-independent but T-cell-dependent mechanisms of immunity (2, 15, 22). Both responses require CD4⁺ αβ T cells; in addition, the expression of cell-mediated immunity (CMI) during both acute and chronic malaria is dependent on γδ T cells activated by CD4⁺ αβ T cells (29, 47, 49, 50). Wild-type (WT) mice depleted of γδ T cells by antibody treatment or gene knockout suppress P. chabaudi parasitemia by antibody-mediated immunity (AMI) (21, 52). Mice depleted of B cells by the same procedures also cure their acute infections in the same timeframe as intact control mice but then develop chronic low-grade parasitemia of long-lasting duration, indicating that B cells and their antibodies are needed to sterilize the infection as we originally reported (15, 48) and has since been confirmed by others (51). B-cell-deficient mice depleted of γδ T cells cannot suppress P. chabaudi parasitemia (49, 50, 52).The prominent role played by IFN-γ in immunity to malaria is generally accepted by most researchers. P. chabaudi malaria is more severe in WT mice treated with neutralizing antibody and in IFN-γ^−/− mice, as indicated by the increased magnitude and duration of parasitemia and mortality in mice deficient in IFN-γ versus intact controls (24, 39, 46). In B-cell-deficient animals, the similar neutralization of IFN-γ by treatment with anti-IFN-γ monoclonal antibody (MAb) or gene knockout of IFN-γ has an even greater effect on the time course of parasitemia, which remains at high levels and fails to cure (1, 46), indicating that IFN-γ is essential for the expression of anti-parasite CMI and contributes to AMI in this model system.The early source of IFN-γ remains controversial, with both NK cells and γδ T cells being proposed to produce this critical cytokine necessary for the activation of the adaptive immune response and the development of protective immunity (9). The results of earlier genetic studies failed to correlate susceptibility to P. chabaudi infection with NK activity (31, 44). Subsequently, Mohan et al. (25) reported that NK cell activity against tumor cell targets correlates with protection against P. chabaudi; anti-asialo GM1 polyclonal antibody depletion of NK cells results in significantly increased levels of peak parasitemia and a prolonged duration of infection compared to controls. The mode of action by which NK cells function appears to be via the secretion of cytokines (25) rather than direct cytotoxicity against the blood-stage parasites. The surface expression of lysosome-associated membrane protein 1 (LAMP-1) by subsets of human NK cells exposed to Plasmodium falciparum-infected erythrocytes may suggest otherwise (20). NK cells in collaboration with dendritic cells are responsible for optimal IFN-γ production dependent upon IL-12 (17, 36, 39, 40). In contrast to the findings of Mohan et al., other studies indicate similar P. chabaudi parasitemia in depleted mice and intact controls after NK1.1 MAb depletion of NK cells (19, 41, 53). Using microarray analysis of blood cells from P. chabaudi-infected mice, Kim et al. (18) reported a rapid production of IFN-γ and activation of IFN-γ-mediated signaling pathways as early as 8 h after infection; however, NK cells did not express IFN-γ or exhibit IFN-γ-mediated pathways in their analysis. At this time, NK cells are replicating and migrating from the spleen to the blood. In humans with P. falciparum malaria, increased production of IFN-γ by PBMC in response to parasitized RBCs correlates with protection from high-density parasitemia and clinical malaria (10, 11); early IFN-γ production by PBMC obtained from malaria naive donors is primarily by γδ T cells and not by NK cells (26). Animal models by definition do not exactly mimic the human condition, and the experimental malaria in mice uses distinct species from those that infect humans. Nevertheless, analysis of protective immunity provides important information on how a protective immune response to Plasmodium may be elicited.Whether both NK cells and γδ T cells have essential roles during the early stages of the immune response to blood-stage malaria remains to be determined. Likewise, whether these cells function early in CMI to malaria parasites is unknown. To address these issues, we infected NK-cell- or γδ-T-cell-depleted J_H^−/− mice with blood-stage P. chabaudi. The resulting time course of parasitemia was monitored and compared to control mice. In addition, spleen cells from depleted and control mice were profiled by cytofluorimetry, and the serum levels of inflammatory cytokines were measured.