Expanded CD14+ CD16+ Monocyte Subpopulation in Patients with Acute and Chronic Infections Undergoing Hemodialysis

Infections are frequent complications in end-stage renal failure patients undergoing hemodialysis (HD), and peripheral blood monocytes are important cells in host defense against infections. The majority of circulating monocytes express high levels of lipopolysaccharide receptor antigen CD14 and are negative for the immunoglobulin Fcγ receptor type III (CD16). We studied the occurrence of a minor subpopulation coexpressing low levels of CD14 together with CD16 in HD patients. In healthy controls CD14⁺ CD16⁺ monocytes account for 8% ± 4% of CD14⁺ monocytes, with an absolute number of 29 ± 14 cells/μl. In stable HD patients the CD14⁺ CD16⁺ subpopulation was significantly elevated (14% ± 3%, or 66 ± 28 cells/μl), while the number of CD14⁺⁺ monocytes (monocytes strongly positive for CD14) remained constant. In HD patients suffering from chronic infections a further rise in CD14⁺ CD16⁺ monocytes was observed (128 ± 71 cells/μl; P < 0.01) such that this subpopulation constituted 24% of all blood monocytes. In contrast, numbers of CD14⁺⁺ cells did not change compared to those for stable HD patients, indicating that the CD14⁺ CD16⁺ monocyte subpopulation was selectively expanded. During acute infections the CD14⁺ CD16⁺ cell subpopulation always expanded. A whole-blood assay revealed that CD14⁺ CD16⁺ monocytes exhibited a higher phagocytosis rate for Escherichia coli bacteria than CD14⁺⁺ monocytes, underlining their role during host defense. In addition, CD14⁺ CD16⁺ monocytes expressed higher levels of major histocompatibility complex (MHC) class II antigens (HLA-DR, -DP, and -DQ) and equal amounts of MHC class I antigens (HLA-ABC). Thus, CD14⁺ CD16⁺ cells constitute a potent phagocytosing and antigen-presenting monocyte subpopulation, which is expanded during acute and chronic infections commonly observed in chronic HD patients.

Peripheral blood monocytes are members of the mononuclear phagocytic system, which plays a central role in immunoregulation and host defense against immunopathogenic organisms (7). Monocytes are activated through molecular signals provided by structures of the infective organisms (8, 27, 28, 34, 35) or inflammatory mediators and chemotactic factors released by other cells during the infective challenge (22, 44, 47). However, blood monocytes represent a heterogeneous cell population and can be distinguished by variations in morphology (38, 58), membrane antigen expression (39), and release of inflammatory mediators (12, 25, 41).While the lipopolysaccharide (LPS) receptor antigen CD14 is expressed by nearly all circulating peripheral blood monocytes, monocytes differ markedly in cell surface CD14 density as well as in the expression of immunoglobulin Fcγ receptors (53, 67). The majority of monocytes strongly positive for CD14 (CD14⁺⁺) express Fcγ receptor I (CD64) and Fcγ receptor II (CD32) and are negative for Fcγ receptor III (CD16) (18). Only a small population was identified by the absence of Fcγ receptors (63). Nevertheless, a subset of monocytes characterized by low-level expression of CD14 and expression of the CD16 antigen has also been described (40). In healthy subjects these CD14⁺ CD16⁺ cells account for about 10% of all monocytes and are thought to be more mature cells than the regular CD14⁺⁺ monocytes, as they exhibit features of tissue macrophages (66). In various infectious or inflammatory diseases such as AIDS and asthma the CD14⁺ CD16⁺ monocyte subpopulation is markedly expanded (36, 43, 50). A more than 10-fold increase of these cells during septicemia was demonstrated, and CD14⁺ CD16⁺ cells become the predominant type of monocytes in some septic patients (14).Patients with end-stage renal failure undergoing chronic hemodialysis (HD) show an impaired immune response (10) with a high prevalence of infectious complications (17). Most of these infections are of bacterial origin, representing a major cause of morbidity and mortality in chronic HD patients (24). Furthermore, acute or chronic inflammatory processes, among them pneumonia and vascular access site infections, are common hazards in uremic patients undergoing chronic regular HD. Despite some data on the functional abnormalities of polymorphonuclear leukocytes in uremia (19), little information exists on the level of monocytes and their subsets in maintenance dialysis patients.In an effort to further understand the importance of the distinct monocyte population expressing Fcγ receptor type III, we determined the levels of these cells in patients with end-stage renal failure undergoing chronic HD. This allowed the level of CD14⁺ CD16⁺ cells to be compared to that of CD14⁺⁺ cells and the total monocyte count in whole blood. To investigate the proinflammatory role of CD14⁺ CD16⁺ monocytes, stable patients as well as patients with acute or chronic signs of infections or inflammatory processes were studied. Furthermore, we analyzed cell surface HLA expression of CD14⁺ CD16⁺ monocytes by immunophenotyping and compared their phagocytic competence with that of regular CD14⁺⁺ blood monocytes.     

CD1 Presents Antigens from a Gram-Negative Bacterium,Haemophilus influenzae Type b

Human CD1 is a family of nonpolymorphic major histocompatibility complex class I-like molecules capable of presenting mycobacterial lipids, including lipoarabinomannan (LAM), to double-negative (DN; CD4⁻ CD8⁻) as well as CD8⁺ T cells. Structural similarities between LAM and the capsular polysaccharides of gram-negative bacteria led us to consider the latter as candidate CD1 ligands. We derived two CD1-restricted DN T-cell populations which proliferated to Haemophilus influenzae type b (Hib) antigen. One T-cell population also proliferated to proteinase K-treated Hib antigen, suggesting that it recognized a nonpeptide. Our work thus expands the universe of T cell antigens to include nonpeptides distinct from mycobacterial lipids and suggests a potential role for CD1-restricted T cells in immunity to Hib.Human CD1 is a family of nonpolymorphic major histocompatibility complex (MHC) class I-like molecules (CD1a to CD1d) (4, 7, 15, 18). Although CD1 is encoded outside the MHC, its association with β2-microglobulin relates it structurally to MHC class I. CD1 molecules are expressed on immature thymocytes (19) and antigen-presenting cells (APC) including cytokine-activated macrophages (13), B cells (22, 23), and dermal dendritic cells (9). Recent studies have revealed that CD1 possesses the unique function of presenting nonpeptide antigen (Ag) to T cells (3, 17, 21, 24). A prototypic Ag presented in the context of CD1 is lipoarabinomannan (LAM), a mannose polymer substituted at one end with arabinose and at the other with a phosphatidic acid containing tubulostearic and palmitic acids. De-O-acylation of LAM totally abrogated T-cell responsiveness, suggesting that the lipid moiety was required for Ag recognition (21). Since gram-negative bacteria contain lipoglycans structurally analogous to LAM (2, 11, 14, 20), we sought to isolate CD1-restricted T cells which recognize antigens from Haemophilus influenzae type b (Hib), a representative gram-negative bacterium.     

Differential Responses of Human Mononuclear Phagocytes to Mycobacterial Lipoarabinomannans: Role of CD14 and the Mannose Receptor

CD14 is a signaling receptor for both gram-negative bacterial lipopolysaccharide (LPS) and mycobacterial lipoarabinomannan (LAM) that lacks terminal mannosyl units (AraLAM). In contrast, terminally mannosylated LAM (ManLAM) binds the macrophage mannose receptor (MMRc), although the ability of the MMRc to serve as a signaling receptor has not been previously reported. We compared the abilities of AraLAM and ManLAM to induce distinct responses in two monocytic cell populations, freshly isolated human peripheral blood monocytes (PBM) and monocyte-derived macrophages (MDM). The responses examined were chemotaxis and transient changes in free cytosolic calcium ([Ca²⁺]_in). We found that AraLAM but not ManLAM was chemotactic for both PBM and MDM. Migration of these cells in vitro to AraLAM was specifically blocked by an anti-CD14 monoclonal antibody, suggesting that CD14 mediates the chemotactic response to AraLAM. Subsequently, we found that AraLAM induced a transient rise in [Ca²⁺]_in levels within a subpopulation of PBM but not MDM. This response was blocked by anti-CD14 antibodies. In contrast, ManLAM induced a transient rise in [Ca²⁺]_in levels within a subpopulation of MDM but not PBM. This response was blocked by either anti-CD14 or anti-MMRc antibodies. These data suggest that the MMRc can serve as a signaling receptor and that coligation of both CD14 and the MMRc is required to elicit a specific response. Thus, one response to LAM (chemotaxis) can be elicited solely by engaging CD14, whereas a different response (changes in [Ca²⁺]_in levels) depends on both the differentiation state of the cells and concomitant engagement of CD14 and the MMRc.Uptake of Mycobacterium tuberculosis by mononuclear phagocytes is the first step leading to the development of tuberculosis infection. Following ingestion of the bacilli, the innate immune response against tuberculosis is predominantly directed by activated macrophages (reviewed in reference 17). The cell wall glycolipid lipoarabinomannan (LAM) is one of many mycobacterial products that can affect these immune responses. Vesicles containing LAM are released from phagosomes following macrophage ingestion of M. tuberculosis (36, 38), suggesting that transport of mycobacterial products out of infected macrophages is possible. Furthermore, the presence of anti-LAM antibodies in the sera of tuberculosis patients suggests that LAM is released from infected macrophages in vivo (29). LAM is comprised of a mannose-rich core polysaccharide, containing highly branched arabinofuranosyl side chains, linked via a phosphatidylinositol moiety at the reducing terminus to acyl groups consisting of palmitic and tuberculostearic acids. LAM isolated from pathogenic M. tuberculosis and M. bovis BCG is capped with mannose residues at the nonreducing arabinofuranosyl termini (ManLAM), whereas LAM isolated from rapidly growing avirulent mycobacteria lacks mannose caps at the arabinofuranosyl ends (AraLAM [10, 26]). The presence or absence of terminal mannose residues has been shown to affect the biological activity of LAM. For example, tumor necrosis factor (TNF) production can be induced in macrophages by purified LAM, although AraLAM is 100-fold more potent in this respect than ManLAM (11, 13). Similar results have been observed for interleukin-1 (IL-1) (41), IL-6 (13), chemokines (28, 40), and nitric oxide (28) production. In contrast, both AraLAM and ManLAM induce similar amounts of transforming growth factor β (TGF-β) production in human monocytes (13).Two potential LAM receptors have been identified on monocytic cells. Zhang and colleagues first showed that the release of IL-1β and TNF by LAM-stimulated human blood mononuclear cells could be blocked by an anti-CD14 monoclonal antibody (MAb) (40). CD14 is a 55-kDa glycosylphosphatidylinositol-linked protein expressed on the surface of monocytes, macrophages, microglial cells, and polymorphonuclear leukocytes which serves as a receptor for gram-negative bacterial lipopolysaccharide (LPS) (reviewed in reference 42). Evidence that LAM can bind directly to CD14 was provided by the demonstration that AraLAM could compete for the binding of LPS to soluble CD14 in vitro (27). A role for CD14 in the receptor-mediated uptake of nonopsonized M. tuberculosis was suggested by studies which showed that both anti-CD14 MAbs and soluble CD14 could significantly block the uptake of M. tuberculosis by human microglial cells (25). In contrast, ManLAM has been shown to function as the ligand which is most likely to mediate uptake of M. tuberculosis via the macrophage mannose receptor (MMRc) on human blood monocyte-derived macrophages (MDM) (31, 32). The MMRc is a 162-kDa glycoprotein expressed in abundance on MDM and tissue macrophages but not on freshly isolated peripheral blood monocytes (PBM) (reviewed in reference 33). A role for ManLAM in the MMRc-mediated adherence of M. tuberculosis to MDM was suggested by the finding that an anti-LAM MAb blocked the binding of M. tuberculosis to MDM by up to 49% (31). A subsequent study revealed that differences in the ability of LAM from different strains of M. tuberculosis to mediate adherence to macrophages and to serve as ligands for the MMRc are not solely determined by the presence of terminal mannosyl units (32).In this study, we compared the capacity of AraLAM and ManLAM to regulate different monocytic cell functions in vitro. We found that purified AraLAM, but not ManLAM, could induce a chemotactic response in human PBM and MDM. Antibody blocking and inhibitor data suggest that CD14 serves as a signaling receptor for AraLAM. This chemotactic response is distinct from the abilities of ManLAM and AraLAM to differentially induce a transient rise in free cytosolic calcium levels in the two cell populations. The capacity of PBM to generate a calcium response upon exposure to AraLAM appears to involve CD14, whereas the capacity of MDM to generate a calcium response following exposure to ManLAM requires engagement of both CD14 and the MMRc. Lastly, exposure of MDM to either AraLAM or ManLAM resulted in the selective down-regulation of the function of complement receptor CR3, although LAM treatment did not affect the level of surface CR3 expression.     

Dual Roles of CD40 on Microbial Containment and the Development of Immunopathology in Response to Persistent Fungal Infection in the Lung

Persistent pulmonary infection with Cryptococcus neoformans in C57BL/6 mice results in chronic inflammation that is characterized by an injurious Th2 immune response. In this study, we performed a comparative analysis of cryptococcal infection in wild-type versus CD40-deficient mice (in a C57BL/6 genetic background) to define two important roles of CD40 in the modulation of fungal clearance as well as Th2-mediated immunopathology. First, CD40 promoted microanatomic containment of the organism within the lung tissue. This protective effect was associated with: i) a late reduction in fungal burden within the lung; ii) a late accumulation of lung leukocytes, including macrophages, CD4⁺ T cells, and CD8+ T cells; iii) both early and late production of tumor necrosis factor-α and interferon-γ by lung leukocytes; and iv) early IFN-γ production at the site of T cell priming in the regional lymph nodes. In the absence of CD40, systemic cryptococcal dissemination was increased, and mice died of central nervous system infection. Second, CD40 promoted pathological changes in the airways, including intraluminal mucus production and subepithelial collagen deposition, but did not alter eosinophil recruitment or the alternative activation of lung macrophages. Collectively, these results demonstrate that CD40 helps limit progressive cryptococcal growth in the lung and protects against lethal central nervous system dissemination. CD40 also promotes some, but not all, elements of Th2-mediated immunopathology in response to persistent fungal infection in the lung.CD40, a 48-kDa type I transmembrane protein and member of the tumor necrosis factor receptor family, is a well-described costimulatory molecule expressed on B cells, dendritic cells (DC), macrophages, basophils, and platelets as well as nonhematopoietic cells including fibroblasts, epithelial, and endothelial cells. The ligand for CD40, known as CD154 or CD40L, is a type II transmembrane protein member of the tumor necrosis factor (TNF) superfamily expressed primarily by activated T cells, B cells, and platelets.^1,2,3 CD40 can be induced on DC, monocytes, and macrophages under inflammatory conditions.^4,5 Signaling via the CD40/CD40L pathway exerts numerous biological effects including: i) increased cytokine expression (especially TNF-α and Th1 cytokines interleukin (IL)-12 and interferon (IFN)-α) and nitric oxide production; ii) upregulation of additional costimulatory molecules (CD80 and CD86) on antigen-presenting cells (APC); iii) enhanced cell survival (particularly of B and T cells, DC, and endothelial cells); iv) Ig isotype switching; and v) somatic hypermutation of Ig.^1,4,5The CD40/CD40L signaling pathway contributes to adaptive Th1 immune responses required to clear Leishmanisa spp.,^6,7,8 Trypanosoma spp.,^6,7,8,9 Shistosoma mansoini,¹⁰ and the fungi Candida albicans¹¹ and Pneumocystis spp.¹² The enhanced production of IFN-γ, TNF-α, and nitric oxide associated with CD40/CD40L signaling is thought to be responsible for this protective effect. However, other studies have suggest that CD40/CD40L signaling is not required for successful host defense against Listeria monocytogenes,^13,14 Toxoplasma gondi,¹⁵ lymphocytic choriomeningitis virus,^16,17 or the fungus Hisoplasma capsulatum.^18,19 In models of Mycobacterium spp. infection, CD40 appears dispensable for clearance of an i.v. infection,^20,21 but essential for clearing the organism in response to aerosolized infection in the lungs.^22,23 Thus, the role of CD40 in antimicrobial host defense varies and depends not only on the specific pathogen but also on the primary site of infection.Cryptococcus neoformans, an opportunistic fungal pathogen acquired through inhalation, causes significant morbidity and mortality primarily in patients with AIDS, lymphoid or hematological malignancies, or patients receiving immunosuppressive therapy secondary to autoimmune disease or organ transplantation.^24,25 Infection in non-immunocompromised patients has been reported.^26,27,28 Murine models of cryptococcal infection in CBA/J or BALB/c mice demonstrate that development of a Th1 antigen-specific immune response characterized by IFN-γ production and classical activation of macrophages is required to eradicate the organism.^{29,30,31,32,33,34,35,36,37,38,39,40} In contrast, a model of persistent cryptococcal infection has been developed using C57BL/6 mice;^{41,42,43,44,45,46,47} this model reflects many features observed in humans diagnosed with allergic bronchopulmonary mycosis.⁴⁸ Specifically, these mice fail to clear the organism from the lung and develop characteristic Th2-mediated immunopathology including: i) tissue eosinophilia; ii) airway hyperreactivity, mucus production, and fibrosis; and iii) alternative macrophage activation associated with YM1 crystal deposition.The molecular mechanisms responsible for the immunopathologic response associated with persistent cryptococcal infection are not clearly defined. These features are abrogated in the absence of IL-4,⁴⁵ whereas more severe Th2-mediated lung injury occurs in the absence of IFN-γ.^29,41 TNF-α exerts a protective effect by enhancing IFN- γ production and the subsequent classical activation of lung macrophages.^31,35,49,50 Lymphocytes are critical mediators of this Th2 response as the pathological features of chronic cryptococcal infection are substantially diminished in CD4 T cell-depleted mice despite no change in fungal clearance.⁴²Although interactions between CD4 T cells and APC are critical determinants of T cell polarization in response to cryptococcal lung infection,^{49,51,52,53,54,55} the contribution of specific costimulatory molecules including the CD40/CD40L signaling pathway has not been fully elucidated. In vitro studies suggest that activation of the CD40/CD40L pathway in response to Cryptococcus promotes IFN-γ production by T cells and TNF-α, and nitric oxide (NO) production by monocytes.⁵⁶ In the absence of CD40L, primary pulmonary infection with a weakly virulent strain of C. neoformans was associated with impaired fungal clearance; however, measurements of immune function at the site of infection in the lung or evidence of systemic fungal dissemination were not evaluated.⁵⁷ The potential to target CD40 therapeutically is highlighted by studies showing that treatment of mice with disseminated or intracerebral cryptococcal infection with an agonist antibody to CD40 in combination with IL-2 improves survival.^58,59 In this study, we used gene-targeted CD40-deficient mice (on a C57BL/6 genetic background), a clinically relevant model, and assessments of immune function and histopathology in the lung to identify two unique roles for the CD40-signaling pathway in response to persistent cryptococcal lung infection.     

Marked Suppression of T Cells by a Benzothiophene Derivative in Patients with Human T-Lymphotropic Virus Type I-Associated Myelopathy/Tropical Spastic Paraparesis

In a search for new anti-autoimmune agents that selectively suppress activation of autoreactive T cells, one such agent, 5-methyl-3-(1-methylethoxy)benzo[b]thiophene-2-carboxamide (CI-959-A), was found to be effective. This compound, which is known to suppress tumor necrosis factor alpha (TNF-α)-induced CD54 expression, inhibited the primary proliferative response of the T cell to antigen (Ag)-presenting cells (APCs) including allogenic dendritic cells (DCs), autologous Epstein-Barr virus-infected B cells, and human T lymphotropic virus type I (HTLV-I)-infected T cells. Autoreactive T cells from patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) spontaneously proliferate in vitro, and their activation is reported to be associated with CD54 expression. The spontaneous proliferation of T cells from patients with HAM/TSP was entirely blocked by CI-959-A. However, in this study, the T-cell proliferation in 15 patients with HAM/TSP was found to depend more extensively on major histocompatibility complex (MHC) class II and CD86 than on CD54 Ags. Since most important APCs for the development of HAM/TSP are DCs and HTLV-I-infected T cells, the effect of CI-959-A on DC generation and on the expression of surface molecules on activated T cells is examined. CI-959-A suppressed recombinant granulocyte-macrophage colony stimulating factor (GM-CSF)- and recombinant interleukin-4-dependent differentiation of DCs from monocytes and inhibited the expression of CD54 and, more extensively, MHC class II and CD86 Ags. CI-959-A showed little toxicity toward lymphoma or HTLV-I-infected T-cell lines or toward monocytes and cultured DCs. These results suggest that CI-959-A might be a potent anti-HAM/TSP agent.Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is thought to be an autoimmune disease induced by HTLV-I infection (8, 9, 24). The T lymphocytes obtained from patients with HAM/TSP patients produce interleukin-2 (IL-2) in vivo and proliferate spontaneously in vitro without any additional stimuli or cytokines (35). This spontaneous proliferation of T lymphocytes (SPL) depends on the interaction of T cells with antigen (Ag)-presenting cells (APCs) such as dendritic cells (DCs) (17, 25) and HTLV-I-infected CD4⁺ T cells (15, 32). The DCs localized in the blood and nonlymphoid organs are considered to be functionally immature, in that they are optimized for the uptake and processing of Ag but not for the initiation of primary T-cell responses. However, after the uptake of Ag and exposure to inflammatory agents including tumor necrosis factor alpha (TNF-α) and IL-1, the DCs undergo a process of maturation and gain the ability to present Ag to T cells for their priming (22, 26). In addition to DCs, HTLV-I-infected CD4⁺ T cells directly stimulate autologous CD4⁺ T cells in a major histocompatibility complex (MHC) class II- and CD86 molecule-dependent fashion (32). Among the T cells stimulated with these APCs, some might cross-react with self Ags and closely associate with the development of HAM/TSP.We have been searching for compounds that inhibit the cellular interaction between APCs and T cells to suppress the activation of autoreactive and Ag-specific T cells. The molecules associated with the APC-T cell interaction may provide an effective target for therapy for autoimmune diseases. Binding of APCs and T cells is initiated by contact of adhesion molecules, such as CD54 and CD11a/CD18, expressed on both cells, and induction of sustained proliferation of T cells requires two independent signals provided by APCs: a T-cell receptor-mediated Ag-specific signal and a signal mediated by costimulatory molecules (CSMs) (10, 20) including CD86 and CD58 Ags (1, 11, 31). Blocking of their tight binding through adhesion molecules or interaction of the CSMs with CSM ligands effectively suppressed the abnormal expansion of disease-associated T cells in vivo and in vitro (19, 30, 32) and sometimes effectively induced a long-term unresponsiveness of T cells to recall stimuli.5-Methyl-3-(1-methylethoxy)benzo[b]thiophene-2-carbox-amide (CI-959-A) is known to inhibit CD54 expression, and its derivative is reported to inhibit casein kinase II (4). In the present study, we found that CI-959-A markedly suppressed SPL in patients with HAM/TSP. Furthermore, the compound suppressed the primary T-cell proliferative response to stimuli provided by various APCs, the differentiation of immature DCs from monocytes and their subsequent maturation, and the induction of expression of MHC class II, CD54, and CD86 Ags on activated CD4⁺ T cells.     

Expression of CD74, the Receptor for Macrophage Migration Inhibitory Factor,in Non-Small Cell Lung Cancer

Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that is overexpressed in lung cancer. The MIF receptor was recently discovered and found to be the invariant chain of the HLA class II molecule, CD74. We hypothesized that the expression of this receptor-ligand pair in lung cancer is associated with the angiogenic activity and level of CXC chemokine expression in human specimens of non-small cell lung cancer. We, therefore, performed immunolocalization of CD74 and compared it with the localization of MIF in non-small cell lung cancer to determine their respective locations, as well as the relationship between the co-expression of MIF-CD74 and angiogenic CXC chemokines with tumor angiogenesis. We found intense CD74 expression by immunohistochemistry in 57 of 70 tumors with minimal to no staining in the remaining 13 tumors. Comparing the localization of CD74 with its putative ligand, MIF, we found that CD74 and MIF were co-expressed in tumors in close proximity, and that co-expression of the MIF-CD74 pair was associated with both higher levels of tumor-associated angiogenic CXC chemokines (ie, the ELR score) and greater vascularity compared with tumors in which MIF-CD74 co-expression was not present. We also found that MIF induced angiogenic CXC chemokine expression in an autocrine manner in vitro, a function that was specifically inhibited by antibodies to CD74.Lung cancer, like all cancers, is characterized by pathological angiogenesis. We have shown that macrophage migration inhibitory factor (MIF) induces expression of angiogenic CXC chemokines by tumor-associated monocytes, and that MIF-dependent expression of angiogenic CXC chemokines is one of several major pathways by which lung cancer tumors induce an angiogenic environment. We and others have shown that MIF is markedly overexpressed in lung cancer^1,2,3 and nearly all other common solid tumors; eg, breast,^4,5,6,7,8 ovarian,^9,10 prostate,^6,7,8 bladder,^11,12 and colon cancer,¹³ as well as melanoma.^14,15 In lung cancer MIF expression is associated with increased production of angiogenic CXC chemokines both in vitro,¹⁶ and in vivo.² High expression of MIF in patients with lung cancer is also associated with a worse prognosis for disease-free and overall survival.^2,3,17 MIF was one of the first cytokines ever to be described,^18,19 yet until it was cloned in 1989,¹⁹ many of its biological activities in health and disease remained unknown. It is a unique cytokine with unusual physicochemical properties, and varied biological properties that, in addition to its role in angiogenesis, include antagonism of p53,^20,21 inhibition of Rb function,^22,23 and activation of Akt.²⁴ Others have shown that MIF can directly induce angiogenesis in vivo as well.²⁵ This combination of properties suggests that MIF may play a pivotal role in tumor biology.Until recently the cell surface receptor for MIF remained unknown. Leng and colleagues²⁶ screened a cDNA library for genes capable of conferring MIF binding and discovered that the invariant chain of the HLA class II peptide (CD74) was the cell surface receptor for MIF.²⁶ Despite the role played by MIF in lung cancer, there are few studies demonstrating a role for CD74 in lung cancer. Two studies examined the expression of CD74 in gastric carcinogenesis induced by Helicobacter pylori, and found that it was associated with poorer overall survival.^27,28 One study done to examine the reactivity of a panel of monoclonal antibodies in 25 lung cancer specimens included the LN2 monoclonal antibody against CD74 and found that this was among a panel of lymphoid antibodies that could distinguish small cell from non-small cell lung cancer (NSCLC).²⁹ This study predated the understanding that CD74 may play a role in MIF signaling.We sought to confirm whether CD74 was present in human lung cancer specimens and reasoned that the distribution of CD74 might yield clues as to the role of MIF in lung cancer. For example, if CD74 is primarily expressed in stromal cells, it would suggest that the effects of MIF in these tumors is primarily through influencing this cell population, whereas CD74 expression primarily on the malignant cells in the tumor would suggest that MIF might be working through antagonism of apoptotic pathways, or by autocrine regulation of angiogenic factor expression. We performed the current study to determine the presence and distribution of CD74 in human lung cancer, to compare the distribution of MIF with its putative receptor CD74, and to determine the relationship of these two factors to angiogenesis in their respective tumors.Our results demonstrate that CD74 is expressed in the majority of lung cancer tumors, with expression being found primarily in stromal compartments in some tumors, whereas others show mixed stromal and malignant epithelial expression. We also observed co-expression of CD74 in close proximity to the ligand MIF and found that CD74 co-expression with MIF was associated with higher levels of angiogenic CXC chemokines and greater tumor vascularity, as measured by factor VIII staining. We further found that in vitro inhibition of MIF or its receptor resulted in reduced production of angiogenic CXC chemokines by human lung cancer cells.     

Modulation of Murine Lyme Borreliosis by Interruption of the B7/CD28 T-Cell Costimulatory Pathway

Recent studies have implicated cytokines associated with Th2 cells in the genetic resistance to murine Lyme borreliosis. Because the B7/CD28 costimulatory pathway has been shown to influence the differentiation of Th-cell subsets, we investigated the contribution of the B7 molecules CD80 and CD86 to the Th2 cytokine profile and development of arthritis in BALB/c mice infected with Borrelia burgdorferi. Effective blockade of CD86/CD28 interaction was demonstrated by elimination of interleukin 4 (IL-4) and upregulation of gamma interferon (IFN-γ) responses by B. burgdorferi-specific T cells and by reduction of B. burgdorferi-specific immunoglobulin G. Despite the shift toward a Th1 cytokine pattern, which others have associated with disease susceptibility, the severity of arthritis was unchanged. Moreover, combined CD80/CD86 blockade by using anti-CD80 and anti-CD86 monoclonal antibodies or CTLA-4Ig enhanced IFN-γ production over that seen with CD86 blockade alone, yet augmentation of this Th1-associated cytokine did not enhance disease. These results demonstrate that IL-4 production by T cells in B. burgdorferi-infected BALB/c mice is dependent upon CD86/CD28 interaction and that this cytokine does not contribute significantly to host resistance to the development of arthritis. In addition, combined CD80/CD86 blockade resulted in preferential expansion of IFN-γ-producing T cells in B. burgdorferi infection, suggesting that costimulatory pathways other than B7/CD28 may contribute to T-cell activation during continuous antigen stimulation. These studies may provide insight into the role of the B7/CD28 pathway in other infectious and autoimmune diseases in which deviation of Th cell immune responses occurs and antigen is persistently present.Lyme disease is a multisystem illness due to infection with the tick-transmitted spirochete Borrelia burgdorferi. Experimental infection of laboratory mice with B. burgdorferi results in acute arthritis and carditis that reproducibly peak at 2 to 4 weeks of infection and then resolve within 3 months despite spirochete persistence (4). Studies using SCID mice, which lack functional T and B cells, have demonstrated that disease is due to the innate immunity of the host and can occur in the absence of specific immune responses (6, 28). The persistent and progressive nature of disease manifestations in SCID mice underscores the importance of T and B cells in initiating disease regression (5, 6, 28). Recent studies support the additional role of specific immunity in modulating disease severity via direct effects on spirochete burden through B. burgdorferi-specific antibodies (5) and indirectly through Th cell-associated cytokines that influence the activation of innate immune cells (14, 23). In particular, the dominance of Th1-type responses, which support macrophage activation, in patients with chronic Lyme arthritis has implicated this T-cell phenotype in the development and perpetuation of severe inflammatory disease (32, 37). Th1-type responses have also been observed during B. burgdorferi infection of C3H mice, a disease-susceptible strain, whereas Th2 responses, which promote B-cell functions, can be detected in BALB/c mice, a comparatively disease-resistant strain (14, 23). Despite the greater inflammatory response in C3H mice, their pathogen burden as assessed by quantitative PCR of spirochete DNA remains higher than that of disease-resistant mouse strains (36), suggesting that the recruitment of innate immune cells is appropriate yet ineffective at controlling infection (29).In addition to signals provided by T-cell antigen receptor engagement, the interaction of costimulatory molecules present on antigen-presenting cells (APCs) with their ligands on T cells is believed to be necessary for the initial priming of naive T cells. In particular, the B7/CD28 costimulatory pathway has been implicated in the differentiation of naive Th0 cells into Th1 and Th2 subsets (33). The mechanisms by which these molecules assist in the priming of the T-cell immune response are complex and poorly understood. Two members of the B7 family have been characterized, CD80 and CD86 (also known as B7-1 and B7-2, respectively), and differ not only in their binding properties to CD28 on T cells but also in the timing of their appearance on conventional APCs during the initiation of an immune response (11). CD86 appears earlier on the surface of mitogen-activated APCs and has a lower affinity for CD28 than does CD80. Once activated, T cells express CTLA-4, a second receptor to which both CD80 and CD86 bind with greater affinity than they bind CD28 (21). Interaction of CD80/CD86 with CTLA-4 can downregulate the T-cell immune response (35). Blockade of CD86 during the initiation of a T-cell response results in an immune response oriented toward a Th1 phenotype, whereas a similar blockade of CD80 does not consistently favor a Th2 phenotype (20). Experiments using mutant mice deficient in CD80 and/or CD86 reveal the important role of these molecules in sustaining a Th-cell phenotype and, in the case of CD86 expression, in the development of a Th2 response (20). Costimulation through the B7/CD28 pathway contributes to the expansion of autoimmune disease processes seen in experimental autoimmune encephalitis (17, 27), a predominantly Th1-associated disease, and autoimmune diabetes (19). Studies using a soluble recombinant form of CTLA-4 designated CTLA-4Ig have supported many of the observations made with anti-B7 antibodies (13, 19, 26).We have recently reported that the Th2 response of B. burgdorferi-infected BALB/c mice is preceded by a Th1 response and that the presence of interleukin 4 (IL-4) is associated with accelerated resolution of arthritis (12). A hind-foot inoculation route was used in that study so that T-cell responses could be examined in lymph nodes adjacent to joints afflicted with arthritis. We demonstrated that this route of inoculation induces moderately severe arthritis in BALB/c mice at day 14 of infection that undergoes more rapid regression than the arthritis seen in similarly infected C3H mice, in which IL-4 responses are not detectable. Previous studies have shown that treatment of mice with anti-IL-4 monoclonal antibody (MAb) exacerbates arthritis in BALB/c mice assessed at intervals corresponding to the plateau and resolution phases of disease, providing evidence that IL-4 modulates the severity of established arthritis (14, 23). The influence of Th2 cell effector functions on the development of arthritis remains unknown. In the current study, we have examined the effects of interruption of Th2 cell differentiation by B7/CD28 blockade with anti-CD80 and/or anti-CD86 MAb or CTLA-4Ig on the cytokine profiles and development of arthritis in BALB/c mice infected with B. burgdorferi.     

Molecular Cloning and Sequencing of Three Granulocytic Ehrlichia Genes Encoding High-Molecular-Weight Immunoreactive Proteins

Granulocytic Ehrlichia was isolated from canine blood obtained from animals challenged with field-collected Ixodes scapularis and propagated in HL60 cells. PCR primers specific for the 16S ribosomal DNA (rDNA) of the Ehrlichia genogroup comprising E. equi, E. phagocytophila, and the agent of human granulocytic ehrlichiosis (HGE) amplified DNA from extracts of these cells. Sequence analysis of this amplified DNA revealed that it is identical to the 16S rDNA sequence of the HGE agent. A genomic library was constructed with DNA from granulocytic Ehrlichia and screened with pooled sera from tick-challenged, granulocytic Ehrlichia-infected dogs. Several clones were isolated and sequenced. Three complete genes encoding proteins with apparent molecular masses of 100, 130, and 160 kDa were found. The recombinant proteins reacted with convalescent-phase sera from dogs and human patients recovering from HGE. This approach will be useful for identifying candidate diagnostic and vaccine antigens for granulocytic ehrlichiosis and aid in the classification of genogroup members.Members of the genus Ehrlichia include species which have a tropism for mononuclear phagocytes (E. canis, E. chaffeensis, E. muris, E. sennetsu, and E. risticii) (33) and those which infect granulocytes (E. ewingii [33], E. phagocytophila [10, 33], E. equi [14, 26], and the recently discovered agent of human granulocytic ehrlichiosis [HGE] [3, 7]). Disease caused by granulocytic Ehrlichia (GE) is manifested by fever, lethargy, thrombocytopenia, and death, and many species from diverse geographical locations have shown evidence of natural infection, including horses (25, 27, 29, 38), dogs (24, 34, 35), small mammals (39, 40), and humans (4, 15).The similar host range and near identity of the 16S rRNA genes of E. phagocytophila, E. equi, and the HGE agent (7) have raised the possibility that these organisms represent a single species (2, 28). In addition, Dumler et al. (9) have shown that they share significant antigenicity by immunofluorescence and immunoblot assays. Objective methods for species classification, e.g., molecular genetic analysis, have not been readily available, primarily because of an inability to culture these ehrlichiae in vitro. However, we (reference 43 and unpublished data) and others (13) have recently demonstrated successful cultivation of GE isolates from dogs and humans, respectively.In this paper, we describe the use of purified GE obtained from in vitro culture of infected HL60 cells, a promyelocytic human cell line, to generate a genomic DNA library for expression screening with sera obtained from dogs experimentally infected with GE. The screening resulted in the isolation of recombinant clones containing complete genes encoding three putative proteins of GE, GE 160, GE 130, and GE 100 (named for apparent molecular mass in kilodaltons). One of these proteins, the 100-kDa protein, is similar in both glutamic acid content and repeated amino acid structure to an immunodominant 120-kDa E. chaffeensis protein (45). Both the 100- and 130-kDa granulocytic Ehrlichia proteins share some amino acid sequence homology to the 120-kDa E. chaffeensis protein.     

Circulating Monocytes Expressing CD31 : Implications for Acute and Chronic Angiogenesis

To identify the roles of various circulating cells (eg, endothelial and/or stem and progenitor cells) in angiogenesis, we parabiosed a wild-type syngeneic mouse with a transgenic syngeneic green fluorescent protein mouse. Following the establishment of a common circulation between these parabionts, we investigated acute (7 to 10 days), subacute (2 to 3 weeks), and chronic (4 to 6 weeks) phases of angiogenesis in wild-type mice using wound healing, implanted gel foam fragments, and subcutaneous tumor assays, respectively. We found that under in vitro conditions, circulating murine monocytes expressed F4/80, CD31, and vascular endothelial growth factor receptor 2, but neither CD133 nor von Willebrand factor, whereas murine endothelial cells expressed CD31, vascular endothelial growth factor receptor 2, and von Willebrand factor, but neither CD133 nor F4/80. Immunofluorescence analysis revealed that green fluorescent protein-positive cells in the walls of new vessels in wounds, gel foam blocks, and tumors expressed both F4/80 and CD31, that is, macrophages. Pericytes, cells that express both CD31 and desmin, were found both in the walls of tumor-associated vessels and within tumors. Collectively, these data demonstrate that monocytes (ie, cells that express both CD31 and F4/80) may be recruited to the site of tissue injury and directly contribute to angiogenesis, reaffirming the close relationships between various cell types within the reticuloendothelial system and suggesting possible targets for anticancer treatments.The progressive growth of neoplasms and establishment of cancer metastasis depend on the development of adequate vasculature, ie, angiogenesis.^1,2,3,4 The identification of critical factors that contribute to angiogenesis is a major goal of antivascular therapy.^5,6 Whether postnatal neovascularization results from the proliferation and migration of endothelial cells of pre-existing blood vessels^7,8,9 or from circulating stem and progenitor cells that are mobilized from the bone marrow and differentiate into mature endothelial cells^{10,11,12,13,14,15} has been controversial. Circulating endothelial cells (CD31+) have been reported to participate in blood vessel formation occurring during physiological and pathological processes, such as inflammation, wound healing, cardiovascular diseases, and cancer,^{9,10,11,12,13,14,15,16,17,18} and these circulating cells have been targets of cancer therapy.^19,20,21,22 While several investigators have concluded that tumor-associated blood vessels consist of 50% bone marrow-derived endothelial cells,^23,24,25 others have reported that the contribution of circulating bone marrow-derived endothelial cells was either low or undetectable.^26,27 One possibility to account for these differences could be the mice under study and their general state of health. In several studies reporting the participation of circulating CD31+ cells in angiogenesis, mice were given lethal x-irradiation or a high dose of chemotherapy leading to myeloablation and then reconstituted with green fluorescence protein (GFP)-labeled bone marrow cells. Whether the same pattern of angiogenesis occurs in physiological conditions is unclear.To directly investigate the contribution of circulating endothelial cells to the establishment of neovasculature in normal mice, we used the same technique that ruled out the assumption that ovulated oocytes in adult mice are derived from circulating germ cells,²⁸ ie, we examined the formation of blood vessels in wounds, implanted gel-foam sponges, and subcutaneous tumors in mice surgically joined by parabiosis.^29,30 Specifically, we joined a genetically marked GFP mouse with a normal mouse. The parabiosed mice developed a common circulation,^28,29,30,31 which allowed us to track genetically marked cells passing from one parabiont to the other and to determine whether these circulating cells contributed to the establishment of blood vessels in healing wounds, gel foam sponges, and subcutaneous tumors.