Neutrophils contribute to development of a protective immune response during onset of infection with Leishmania donovani

Neutrophils are key components of the inflammatory response and as such contribute to the killing of microorganisms. In addition, recent evidence suggests their involvement in the development of the immune response. The role of neutrophils during the first weeks post-infection with Leishmania donovani was investigated in this study. When L. donovani-infected mice were selectively depleted of neutrophils with the NIMP-R14 monoclonal antibody, a significant increase in parasite numbers was observed in the spleen and bone marrow and to a lesser extent in the liver. Increased susceptibility was associated with enhanced splenomegally, a delay in the maturation of hepatic granulomas, and a decrease in inducible nitric oxide synthase expression within granulomas. In the spleen, neutrophil depletion was associated with a significant increase in interleukin 4 (IL-4) and IL-10 levels and reduced gamma interferon secretion by CD4⁺ and CD8⁺ T cells. Increased production of serum IL-4 and IL-10 and higher levels of Leishmania-specific immunoglobulin G1 (IgG1) versus IgG2a revealed the preferential induction of Th2 responses in neutrophil-depleted mice. Altogether, these data suggest a critical role for neutrophils in the early protective response against L. donovani, both as effector cells involved in the killing of the parasites and as significant players influencing the development of a protective Th1 immune response.     

Differential expression of mycobacterial antigen MPT64, apoptosis and inflammatory markers in multinucleated giant cells and epithelioid cells in granulomas caused by <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

The development of granulomas is a major histopathological feature of tuberculosis. Very little information is available concerning the physiology and functions of different cell types in the tuberculous granulomas. The aim of this study was to compare the epithelioid cells (ECs) and multinucleated giant cells (MGCs) in the granulomas caused by Mycobacterium tuberculosis complex organisms. Lymph node biopsies from 30 cases of lymphadenitis were studied for expression of the secreted mycobacterial protein MPT64, caspase 3 as a marker of apoptosis, apoptosis-related proteins (Fas Ligand, Fas and Bax) and inflammatory cytokines (interleukin-10, transforming growth factor-β (TGF-β), tumour necrosis factor-α and interferon-γ) by immunohistochemistry. MGCs more often contained M. tuberculosis secretory antigen MPT64 (p < 0.001) and expressed more TGF-β (p = 0.004) than ECs. The total number of apoptotic MGCs was higher than the number of apoptotic ECs (p = 0.04). Interestingly, there was a significant negative correlation between apoptosis and MPT64 expression in MGCs (r = −0.569, p = 0.003), but not in ECs, implying that the heavy antigen load would lead to inhibition of apoptosis in these cells. When compared with ECs, higher percentage of MGCs expressed Fas Ligand and Fas (p < 0.004). The role of MGCs may thus be different from surrounding ECs and these cells by virtue of higher mycobacterial antigen load, more TGF-β and reduced apoptosis may contribute towards persistence of infection.     

Matrix metalloproteinases and their tissue inhibitors in the lesions of cardiac and pulmonary sarcoidosis: an immunohistochemical study

The pathogenesis of the tissue damage and fibrosis in sarcoidosis is poorly understood. The matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) must be considered in this regard, because they control the lysis of connective tissue components. Immunohistochemical studies (peroxidase and dual labeling for confocal microscopy) of reactivity for MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and the 4 membrane-type-MMPs were made on tissues from patients with cardiac (n = 4) and pulmonary (n = 5) sarcoidosis. The granulomas were histochemically similar in both organs. The multinucleated giant cells (MGCs) showed moderate reactivity for MMP-1 and MMP-9 and variable reactivity for MMP-2 and MMP-3; in addition, they showed colocalization of MT-1-MMP, which activates MMP-2. The reactivity of epithelioid cells (ECs) was moderate for MMP-2 and mild for other MMPs. Macrophages showed weaker reactivity for MMPs than did MGCs and ECs. All 3 types of cells showed very low reactivity for TIMPs. Staining for type IV collagen showed focal damage to the basement membranes of cardiac myocytes and pulmonary alveoli near the granulomas. The cells in sarcoid granulomas contain an abundance of MMPs and a paucity of TIMPs. The MGCs also contain MT-1-MMP and thus can activate MMP-2 in the granulomas. The MMPs can cause damage to adjacent cardiac myocytes and pulmonary alveoli, leading to the interstitial fibrosis produced by sarcoidosis.     

Transforming Growth Factor β and Immunosuppression in Experimental Visceral Leishmaniasis

Hamsters infected with Leishmania donovani develop a disease similar to human kala-azar. They present hypergammaglobulinemia, and their T cells do not respond to parasite antigens. This unresponsiveness has been primarily ascribed to defects in antigen-presenting cells (APCs), because these cells are unable to stimulate proliferation of parasite-specific T cells from immunized animals. In this study, we show that APCs (adherent spleen cells) from L. donovani-infected hamsters produce high levels of the inhibitory cytokine transforming growth factor β (TGF-β). Immunohistochemical studies with an anti-TGF-β monoclonal antibody (MAb) showed that this cytokine is abundantly produced in vivo by the spleen cells of infected animals. In addition, high levels of TGF-β are produced in vitro by infected hamster cells, either spontaneously or after stimulation with parasite antigen or lipopolysaccharide. Furthermore, in vivo-infected adherent cells obtained from spleens of L. donovani-infected hamsters caused profound inhibition of the in vitro antigen-induced proliferative response of lymph node cells from hamsters immunized with leishmanial antigens. Moreover, this inhibition was totally abrogated by the anti-TGF-β MAb. These results suggest that the immunosuppression observed in visceral leishmaniasis is, at least in part, due to the abundant production of TGF-β during the course of the infection.     

Leishmania donovani infection initiates T cell-independent chemokine responses,which are subsequently amplified in a T cell-dependent manner

Control of Leishmania donovani infection in immunocompetent mice is associated with hepatic inflammation and granuloma formation, both of which are absent in severe combined immunodeficient ( scid ) mice. In both BALB/c and scid mice, L. donovani infection induced a rapid hepatic accumulation of mRNA encoding macrophage inflammatory protein-1α (MIP-1α), monocyte chemoattractant protein-1 (MCP-1) and interferon-γ inducible protein-10 (γIP-10). This response was not preceded by increased IL-4 production in either strain, unlike that reported in other infectious disease models. Interestingly, only γIP-10 mRNA was maintained at elevated levels throughout the first 7 days of infection, by mechanisms involving CD4⁺ and CD8⁺ T cells, and CD4⁻ CD8⁻ cells not activated in scid mice. By in vivo depletion and reconstitution of scid mice it was demonstrated that T cells regulate the expression of all three chemokines studied, while they themselves only produce γIP-10 in appreciable quantities.     

Leishmania donovani‐induced expression of signal regulatory protein α on Kupffer cells enhances hepatic invariant NKT‐cell activation

Signal regulatory protein α (SIRPα) and its cognate ligand CD47 have been documented to have a broad range of cellular functions in development and immunity. Here, we investigated the role of SIRPα–CD47 signalling in invariant NKT (iNKT) cell responses. We found that CD47 was required for the optimal production of IFN‐γ from splenic iNKT cells following exposure to the αGalCer analogue PBS‐57 and in vivo infection of mice with Leishmania donovani. Surprisingly, although SIRPα was undetectable in the liver of uninfected mice, the hepatic iNKT‐cell response to infection was also impaired in CD47^?/? mice. However, we found that SIRPα was rapidly induced on Kupffer cells following L. donovani infection, via a mechanism involving G‐protein‐coupled receptors. Thus, we describe a novel amplification pathway affecting cytokine production by hepatic iNKT cells, which may facilitate the breakdown of hepatic tolerance after infection.     

Asteroid bodies and calcium oxalate crystals: two infrequent findings in fine-needle aspirates of parotid sarcoidosis.

We have studied 3 cases of sarcoidosis involving the parotid gland by means of fine-needle aspiration cytology (FNAC). The main findings were noncaseating granulomas, multinucleated giant cells (MGCs), and lymphocytes. In one case MGCs contained asteroid bodies and in another case we observed calcium oxalate crystals (COCs) over both stromal fragments and MGCs. Although nonpathognomonic for sarcoidosis, these 2 findings may help in the diagnosis of this condition. However, both are easily overlooked and must be borne in mind when viewing noncaseating granulomas. Sarcoid granulomas displaying COC must be differentiated from foreign-body granulomas. The aforementioned cytological findings must be assessed in conjunction with clinical findings. Nevertheless, in most cases the diagnosis of sarcoidosis is made by exclusion.     

Immunoprotective responses of T helper type 1 stimulatory protein‐S‐adenosyl‐L‐homocysteine hydrolase against experimental visceral leishmaniasis

It is well known that a patient in clinical remission of visceral leishmaniasis (VL) remains immune to reinfection, which provides a rationale for the feasibility of a vaccine against this deadly disease. In earlier studies, observation of significant cellular responses in treated Leishmania patients as well as in hamsters against leishmanial antigens from different fractions led to its further proteomic characterization, wherein S‐adenosyl‐L‐homocysteine hydrolase (AdoHcy) was identified as a helper type 1 (Th1) stimulatory protein. The present study includes immunological characterization of this protein, its cellular responses [lymphoproliferation, nitric oxide (NO) production and cytokine responses] in treated Leishmania‐infected hamsters and patients as well as prophylactic efficacy against Leishmania challenge in hamsters and the immune responses generated thereof. Significantly higher cellular responses were noticed against recombinant L. donovani S‐adenosyl‐L‐homocysteine hydrolase (rLdAdoHcy) compared to soluble L. donovani antigen in treated samples. Moreover, stimulation of peripheral blood mononuclear cells with rLdAdoHcy up‐regulated the levels of interferon (IFN)‐γ, interleukin (IL)?12 and down‐regulated IL‐10. Furthermore, vaccination with rLdAdoHcy generated perceptible delayed‐type hypersensitivity response and exerted considerably good prophylactic efficacy (～70% inhibition) against L. donovani challenge. The efficacy was confirmed by the increased expression levels of inducible NO synthase and Th1‐type cytokines, IFN‐γ and IL‐12 and down‐regulation of IL‐4, IL‐10 and transforming growth factor (TGF)‐β. The results indicate the potentiality of rLdAdoHcy protein as a suitable vaccine candidate against VL.     

Muramyl dipeptide and mononuclear cell supernatant induce Langhans-type cells from human monocytes

Muramyl dipeptide (MDP) in bacterial cell walls reportedly evokes epithelioid cell granulomas. We examined its effects on multinucleated-giant-cell (MGC) formation from monocytes. Supernatant of concanavalin A-stimulated peripheral blood mononuclear cells (conditioned medium) generated MGCs from monocytes. MDP significantly increased the fusion index of Langhans-type MGCs (LGCs) but did not affect total MGCs. N-Acetylmuramyl-L-alanyl-L-isoglutamine, an MDP analogue, had no effect on MGC formation. MGCs were produced by conditioned medium from CD14(++)/CD16(-) monocytes. MDP enhanced the LGC fusion index from CD14(++)/CD16(-) monocytes. MGCs were not produced from CD14(+)/CD16(+) monocytes or immature dendritic cells induced by granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL) 4 and only weakly produced from macrophage (M)-CSF- or GM-CSF-induced macrophages. Added MDP did not generate MGCs from CD14(+)/CD16(+) monocytes or dendritic cells but enhanced LGC formation from macrophages. Because IFN-gamma, IL-3, and GM-CSF reportedly are important in LGC induction, we added anti-IFN-gamma, anti-IL-3, or anti-GM-CSF monoclonal antibody (mAb) concomitantly to the monocyte culture treated with conditioned medium alone or plus MDP. Anti-IFN-gamma mAb completely abrogated MGC generation, whereas anti-GM-CSF and anti-IL-3 mAbs significantly inhibited LGCs. These findings suggest that CD14(++)/CD16(-) monocytes are fused to form LGCs by MDP derived from granulomatous-disease-causing pathogens with inflammatory mediators such as IFN-gamma, IL-3, and GM-CSF.     

Neutralization of IL-12 demonstrates the existence of discrete organ-specific phases in the control of Leishmania donovani

IL-12 plays a key role in stimulating both innate and antigen-specific immune responses against a number of intracellular pathogens. A neutralizing anti-IL-12 monoclonal antibody (mAb) was used to define and compare the role of endogenous IL-12 in the liver and spleen of mice infected with Leishmania donovani. IL-12 neutralization both early and late in infection caused delayed resolution of parasite load, a transient decrease in IFN-γ, IL-4, TNF-α and inducible nitric oxide synthase (NOS-2) production, and suppressed tissue granuloma formation in the liver of genetically susceptible BALB/c mice. In contrast to the liver of BALB/c mice, neutralization of IL-12 had no effect on parasite burden in the spleen over the first 28 days of infection. However, IL-12 appeared to be critical for the development of mechanisms which subsequently contain the growth of persistent parasites in this organ in that neutralization of IL-12 dramatically enhanced parasite growth after day 28 of infection. Following IL-12 neutralization, the later unchecked growth of parasites in the spleen was coincident with an extensive breakdown of the tissue microarchitecture. Immunohistochemical studies revealed that IL-12 was largely produced by uninfected cells in L. donovani-infected BALB/c mice. In contrast, the course of infection in the liver and spleen of genetically resistant CBA/n mice was unaffected by the administration of anti-IL-12 mAb. These results suggest that the liver and spleen in susceptible BALB/c mice have different temporal requirements for IL-12 in controlling L. donovani infection, whereas IL-12 plays little role in either organ in resistant CBA/n mice. In addition, IL-12 appears to be involved in the generation of both Th1 and Th2 responses during L. donovani infection in BALB/c mice.     

Autonomous growth of lymphoid cells following IL-2 expression from retrovirus vectors containing HIV-1trans-acting elements

The human immunodeficiency virus type I (HIV-1) possesses powerful regulatory elements that control the rate of replication of HIV-1 and subsequent processing ofHIV-1 genes. We have used this regulatory mechanism to drive expression of foreign genes inserted in retrovirus vectors. This approach was used to express the humanIL-2 gene in IL-2-dependent mouse CTLL-2 cells to determine the role of autonomous growth in maintaining proliferation of virus-infected T lymphocytes during HTLV-1-induced adult T-cell leukemia (ATL). Expression of IL-2 sequences in IL-2-dependent mouse CTLL-2 cells resulted in autonomous growth of IL-2-independent CTLL-2 clones. Endogenous expression of IL-2 appeared to interrupt normal constraints of growth in that these IL-2-independent clones showed reduced cell-density-dependent inhibition but not a tumorigenic phenotype. IL-2-independent CTLL-2 clones did not secrete detectable quantities of IL-2 into culture supernatant and exhibited reduced sensitivity to the inhibitory effects of both IL-2 and IL-2 receptor antibody. These results suggest that the IL-2 autocrine loop within these cells involves intracellular IL-2/IL-2 receptor binding. The apparent lack of IL-2 production and poor responsiveness to IL-2 or IL-2 antibodies displayed by cell lines from ATL patients may be explained by an intracellular IL-2/IL-2 receptor autocrine loop.     

Non-neoplastic pulmonary multinucleate giant cells. Comparative study of multinucleate giant cells in porcine dermatosis vegetans and other polykaryocytic conditions

Pulmonary multinucleate giant cells (MGCs) in porcine dermatosis vegetans (DV) were compared with virus-induced and inflammatory-associated MGCs, with particular attention to mechanisms involved in the formation of such cells. Ultrastructurally, pigs with DV showed numerous pulmonary MGCs with transversely-sectioned interdigitating microvillous structures. Microvilli were observed closely apposed to type-II pneumocytes and with morphological characteristics of membrane bridge formation. Rabbits infected with Mycobacterium bovis exhibited multiple pulmonary granulomas with prominent MGC formation 14 days after infection. Close apposition between MGCs and small macrophages and membrane bridge formation were demonstrated ultrastructurally. Pulmonary changes in calves infected with bovine respiratory syncytial virus (BRSV) included lobular consolidation and intense infiltration of inflammatory cells into the bronchial, bronchiolar and alveolar walls. Proliferation of bronchiolo-alveolar epithelial cells was prominent, but syncytiae were only occasionally demonstrated. This study of the morphological characteristics of MGCs is related to the present knowledge of mechanisms involved in membrane fusion and allows for an interpretation of an active fusion process involved in the formation of MGCs in porcine DV.     

Ribosomes ofLeishmania are a target for the aminoglycosides

Ribosomes ofLeishmania, a parasitic protozoan (member of the order of Kinetoplastidae), were purified on a sucrose density gradient. Two different types of ribosomes were isolated from the promastigotes: cytoplasmic (88S and 91S fromL. tropica andL. donovani, respectively) and mitochondrial (75S in both species). Both types of ribosome dissociated into their subunits at low Mg²⁺ concentration (1–2 mM) as follows: 67S and 49S for the 91S cytoplasmic ribosomes ofL. donovani and 61S and 43S for the 88S cytoplasmic ribosome ofL. tropica; 55S and 34S forL. tropica and 60S and 39S forL. donovani mitochondrial ribosomes, respectively. Paromomycin (aminosidine), an aminoglycoside aminocyclitol antibiotic, interacted with the ribosomes to promote the association of the subunits. Under similar experimental conditions, spermidine and pentamidine were inactive.     

Anti-L3T4 antibody treatment suppresses hepatic granuloma formation and abrogates antigen-induced interleukin-2 production in Schistosoma mansoni infection

In murine schistosomiasis mansoni, granulomatous inflammation is an immune response that involves egg antigen presentation to T cells in the context of class II major histocompatibility complex determinants and subsequent inflammatory lymphokine production by delayed-hypersensitivity (TDH) lymphocytes. In the present study, monoclonal antibodies directed against L3T4, I-A, and Lyt-2 molecules were injected intraperitoneally into S. mansoni-infected mice to study the role of these membrane antigens in the process of granuloma formation. A dramatic suppression of the hepatic granuloma size and antigen-induced interleukin-2 (IL-2) production by spleen cells was seen in mice that received anti-L3T4 monoclonal antibody treatment. The total number of cells, especially the L3T4+ T cells, was greatly diminished in the spleens. Furthermore, histopathological study of the granulomas in stained liver sections demonstrated the paucity of eosinophils and macrophages, absence of epithelioid cells and multinucleated giant cells, and minimal collagen deposition within the lesions. Damaged hepatocytes were also seen surrounding these ill-formed granulomas. In contrast, anti-I-A monoclonal antibody treatment partially suppressed IL-2 production, although granuloma size and cellular composition remained the same. Mice that received anti-Lyt-2 monoclonal antibody did not show any changes in either IL-2 production or hepatic granulomatous inflammation. The data presented in this paper indicate a crucial role for L3T4 molecules present on a subset of class II major histocompatibility complex-restricted TDH cells in IL-2 production and the generation of the granulomatous response.     

Photodynamic vaccination of hamsters with inducible suicidal mutants of Leishmania amazonensis elicits immunity against visceral leishmaniasis

Leishmania, naturally residing in the phagolysosomes of macrophages, is a suitable carrier for vaccine delivery. Genetic complementation of these trypanosomatid protozoa to partially rectify their defective heme‐biosynthesis renders them inducible with δ‐aminolevulinate to develop porphyria for selective photolysis, leaving infected host cells unscathed. Delivery of released “vaccines” to antigen‐presenting cells is thus expected to enhance immune response, while their self‐destruction presents added advantages of safety. Such suicidal L. amazonensis was found to confer immunoprophylaxis and immunotherapy on hamsters against L. donovani. Neither heat‐killed nor live parasites without suicidal induction were effective. Photodynamic vaccination of hamsters with the suicidal mutants reduced the parasite loads by 99% and suppressed the development of disease. These suppressions were accompanied by an increase in Leishmania‐specific delayed‐type hypersensitivity and lymphoproliferation as well as in the levels of splenic iNOS, IFN‐γ, and IL‐12 expressions and of Leishmania‐specific IgG2 in the serum. Moreover, a single intravenous administration of T cells from vaccinated hamsters was shown to confer on naïve animals an effective cellular immunity against L. donovani challenges. The absence of lesion development at vaccination sites and parasites in the draining lymphnodes, spleen and liver further indicates that the suicidal mutants provide a safe platform for vaccine delivery against experimental visceral leishmaniasis.     

Histamine protects against the acute phase of experimentally-induced hepatic ischemia/re-perfusion

Histamine, involved in many inflammatory reactions and immune responses, is reported to suppress—via H4R stimulation—injury concomitant with the late phase of warm hepatic ischemia/re-perfusion (I/R). The current study investigated the possible effects of histamine on the acute phase of hepatic I/R injury, and the possible underlying mechanisms like oxidative stress and release of inflammatory cytokines (e.g., tumor necrosis factor (TNF)-α nd interleukin [IL]-12). Rats were divided into naïve, sham-operated, and I/R groups. The I/R group was divided into sub-groups and pre-treated with histaminergic ligands before induction of ischemia. Anesthetized rats were subjected to warm ischemia for 30 min by occlusion of the portal vein and hepatic artery, then re-perfused for 90 min. Rats in the control I/R group showed significant increases in hepatic malondialdehyde (MDA), TNFα, and IL-12 contents, and in plasma alanine transaminase (ALT) and aspartate transaminase (AST) levels, along with significant decreases in hepatic reduced glutathione (GSH) content and marked diffuse histopathologic damage. Pre-treatment with histamine resulted in significant mitigation of each of these end-points. The protective effect of histamine was not antagonized by pre-treatment with mepyramine (H1R antagonist) or ranitidine (H2R antagonist) and completely reversed by pre-treatment with thioperamide (H3R and H4R antagonist). In addition, the histamine protective effect was mimicked by pre-treatment of rats with clozapine (H4R agonist). These observations strongly suggested that histamine has a protective effect against hepatic I/R-mediated tissue injury during the acute phase, and this effect was mediated through an H4R stimulation that led to a decrease in IL-12 and TNFα production—outcomes that consequently decreased localized oxidative stress and afforded hepatic protection in general.     

Carcinoembryonic antigen promotes tumor cell survival in liver through an IL-10-dependent pathway

Most circulating tumor cells die within 24 h of entering the hepatic microvasculature because their arrest initiates an ischemia-reperfusion (I/R) injury that is cytotoxic. Human colorectal carcinomas (CRC) produce the glycoprotein Carcinoembryonic Antigen (CEA) that increases experimental liver metastasis in nude mice. Since CEA induces release of IL-6 and IL-10, we hypothesized that CEA inhibits the I/R injury through a Kupffer cell-mediated cytokine-dependent pathway. We assessed cytokine effects in CRC co-cultured with liver and in vivo. Human CRC prelabeled with fluorescent dyes were incubated with a reoxygenated suspension of ischemic nude mouse liver fragments in a bioreactor. CEA, rhIL-6 or rhIL-10 were either administered to the donor mice prior to hepatic ischemia or during co-culture. Liver donors were athymic nude or iNOS, IL-6 or IL-10 knock out mice. Ischemic-reoxygenated liver kills Clone A CRC through production of nitric oxide (NO) and superoxide anion. Treatment of liver donors with CEA prior to hepatic ischemia inhibited this in vitro cytotoxicity through an IL-10 and Kupffer cell dependent pathway that inhibited NF-κB activation, NO production and iNOS upregulation. IL-10 but not IL-6 enhanced CRC survival in nude mouse liver in vivo. Thus, CEA enhanced metastasis by inducing IL-10 to inhibit iNOS upregulation in host liver.This revised version was published online in August 2005 with a corrected cover date.