The adapter protein LAT enhances fcgamma receptor-mediated signal transduction in myeloid cells

FcgammaR clustering in monocytes initiates a cascade of signaling events that culminate in biological responses such as phagocytosis, production of inflammatory cytokines, and generation of reactive oxygen species. We have identified and determined the function of the adapter protein linker of activation of T cell (LAT) in FcgammaR-mediated signaling and function. Clustering of FcgammaRs on the human monocytic cell line, THP-1, induces phosphorylation of a major 36-kDa protein which immunoreacts with anti-LAT antisera. Our data indicate that although both the 36-kDa and 38-kDa isoforms of LAT are expressed in THP-1 and U937 human monocytic cells, FcgammaR clustering induces phosphorylation of the 36-kDa isoform only. Co-immunoprecipitation experiments revealed a constitutive association of p36 LAT with both FcgammaRI and FcgammaRIIa immunoprecipitates, and an activation-induced association of LAT with PLCgamma1, Grb2, and the p85 subunit of phosphatidylinositol 3-kinase. Transient transfection experiments in COS-7 cells indicated that overexpression of a wild type but not a dominant-negative LAT, that is incapable of binding to p85, enhances phagocytosis by FcgammaRI. Furthermore, bone marrow-derived macrophages from LAT-deficient mice displayed reduced phagocytic efficiency in comparison to the macrophages from wild-type mice. Thus, we conclude that p36 LAT serves to enhance FcgammaR-induced signal transduction in myeloid cells.     

Regulation of monocyte survival in vitro by deposited IgG: role of macrophage colony-stimulating factor

IgG deposition at tissue sites characteristically leads to macrophage accumulation and organ injury. Although the mechanism by which deposited IgG induces tissue injury is not known, we have recently demonstrated that deposited IgG stimulates the release of IL-8 and monocyte chemoattractant protein-1 from normal human monocytes, which may drive inflammation. Since IgG also induces macrophage accumulation in these diseases, we hypothesized that deposited IgG protects monocytes from apoptosis. As an in vitro model of the effect of deposited IgG on monocyte survival, monocyte apoptosis was studied after FcgammaR cross-linking. Monocytes cultured on immobilized IgG, which induces FcgammaR cross-linking, were protected from apoptosis, whereas monocytes cultured with equivalent concentrations of F(ab')2 IgG or 50 times higher concentrations of soluble IgG, neither of which induces FcgammaR cross-linking, were not protected. Moreover, this protection was transferable, as supernatants from immobilized IgG-stimulated monocytes protected freshly isolated monocytes from apoptosis and contained functional M-CSF, a known monocyte survival factor. M-CSF mediated the monocyte survival induced by FcgammaR cross-linking, as neutralizing anti-human M-CSF Abs blocked the monocyte protection provided by either immobilized IgG or IgG-stimulated monocyte supernatants. These findings demonstrate a novel mechanism by which deposited IgG targets tissue macrophage accumulation through FcgammaR-mediated M-CSF release. This pathway may play an important role in promoting and potentiating IgG-mediated tissue injury.     

A codominant role of Fc gamma RI/III and C5aR in the reverse Arthus reaction

Recent attempts to specify the relative contribution of FcR and complement in various experimental systems of immune complex disease have led to opposing conclusions. As concluded in IgG FcRgamma-/- mice, manifestation of disease is almost exclusively determined by FcgammaR on effector cells, arguing for a minor role of complement. In contrast, data obtained with C5aR-/- mice suggested that, dependent on the tissue site, complement is more important than FcgammaR. In this paper, we demonstrate that, in response to IgG immune complex formation, FcgammaRI/III- and C5aR-mediated pathways are both necessary and only together are they sufficient to trigger the full expression of inflammation in skin and lung. Moreover, both effector systems are not entirely independent, suggesting an interaction between FcgammaR and C5aR. Therefore, FcgammaR-mediated responses can be integrated through C5aR activation, which may explain why these two receptor pathways have previously been considered to dominate each other.     

Regulated expression and inhibitory function of Fcgamma RIIb in human monocytic cells.

Human monocytes/macrophages express three classes of receptors for IgG: FcgammaRI, FcgammaRII, and FcgammaRIII. The expression and function of these receptors has been extensively studied with the exception of one, FcgammaRIIb. While the mRNA for FcgammaRIIb has been detected in human monocytes, the protein has remained elusive. Studies in mouse models indicated that the macrophage FcgammaRIIb serves to down-regulate FcgammaR-mediated phagocytosis and immune complex-induced inflammation. FcgammaRIIb has also been shown to modulate the action of cytotoxic antibodies against tumors in mouse models. Hence, an understanding of how FcgammaRIIb expression is regulated is of great importance. Here we demonstrate for the first time FcgammaRIIb protein expression and function in human monocytes. We also report that the expression of FcgammaRIIb is highly up-regulated by interleukin-4, a Th2 cytokine, and that the up-regulation of FcgammaRIIb results in a decrease in the phagocytic efficiency of interleukin-4-treated THP-1 cells. Furthermore co-clustering FcgammaRIIb with FcgammaRIIa resulted in enhanced phosphorylation of the inositol phosphatase SHIP, association of SHIP with Shc, and phosphorylation of additional proteins around 120 and 60-65 kDa, with a concomitant attenuation of Akt activation. We, therefore, propose that FcgammaRIIb serves to inhibit FcgammaRI/IIa-mediated macrophage activation using SHIP as its effector.     

Transforming growth factor-beta: an important mediator of immunoregulation

Transforming growth factor-beta (TGF-beta) is synthesized and secreted by a wide variety of cells, including cells of the immune system. Lymphocytes and monocytes possess high affinity TGF-beta receptors and the addition of TGF-beta to in vitro cell cultures results in significant modulation of immune function. TGF-beta inhibits the proliferation of thymocytes, T cells, B cells, and natural killer cells. Additionally, it inhibits certain differentiative functions of lymphocytes including a marked inhibition of immunoglobulin production by human B lymphocytes. TGF-beta has dichotomous actions on monocytes. It is a potent chemoattractant for monocytes and induces interleukin 1 mRNA expression while inhibiting generation of reactive oxygen intermediates and monocyte killing. Evidence is accumulating that TGF-beta regulates immune function in vivo and that overproduction of TGF-beta may be associated with immunosuppression.     

Docetaxel promotes the generation of anti-tumorigenic human macrophages

The taxanes Docetaxel and Paclitaxel are two of the standard chemotherapies for patients with metastatic breast cancer. The functional effect of Docetaxel and Paclitaxel on human innate immune cells of the myeloid lineage is not well established, nor is the effects these agents have on differentiation of monocytes into macrophages and dendritic cells. Therefore, the aim with this project was to determine the effects of Docetaxel and Paclitaxel on primary human monocyte differentiation, activation and function. For this purpose, primary human monocytes were isolated from healthy donors and cultured with or without Docetaxel and Paclitaxel. We found that Docetaxel promoted the differentiation of primary human monocytes into pro-inflammatory macrophages with an M1 phenotype and an ability to present antigens to T cells. Monocytes treated with Docetaxel also displayed an elevated secretion of IL-8 and IL-1β, but did not promote generation of monocytic myeloid-derived suppressor cells. In conclusion, Docetaxel appears to have an immune stimulatory effect that would be beneficial for an anti-tumorigenic type of immune response, whereas Paclitaxel seems to have less effect on myeloid cells.     

Effects of transforming growth factor-beta on long-term human cord blood monocyte cultures.

Transforming growth factor-beta (TGF-beta) modulates growth and differentiation in many cell types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF-beta has been shown to influence bone resorption in organ culture, we have studied the effect of TGF-beta (1-1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10(-9) M 1,25(OH)2D3. TGF-beta, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF-beta of 1 ng/ml, the multinucleated cells were reduced to 2.1% +/- 0.3%, compared to 19.3% +/- 1.5% in control cultures. TGF-beta inhibited in a dose-dependent manner the proliferation of cord blood monocytes as assessed by 3H-thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF-beta. Indomethacin did not reverse the inhibitory effects of TGF-beta. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteoclasts but not by adult monocytes, and an antibody to HLA-DR, which is not present on osteoclast. TGF-beta decreased the expression of HLA-DR and increased in a dose-dependent manner the proportion of 23C6-labeled cells; these results suggest that TGF-beta could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF-beta did not induce any 45Ca release from bone cultured with monocytes, suggesting that full osteoclastic differentiation was not achieved. These results emphasize the complex role of TGF-beta in the local regulation of bone cell differentiation and in bone remodeling.     

Regulated expression of FcgammaR in human dendritic cells controls cross-presentation of antigen-antibody complexes

Receptors for IgG (FcgammaR) expressed in dendritic cells (DCs) influence the initiation of Ab-mediated immunity. Dynamic variations in FcgammaR expression allow DCs to adjust their capacity to capture Ab-opsonized Ag. The current paradigm predicts a progressive decline in FcgammaR-mediated phagocytic function upon DC maturation. Surprisingly, we find that expression of the phagocytic receptor FcgammaRIIa is preserved in immature and mature DCs at comparable levels with macrophages. Moreover, phagocytosis of antigenic peptides directed to FcgammaRIIa on DCs leads to dramatic increases in Ag cross-presentation and T cell activation. In immature DCs, high expression of inhibitory FcgammaRIIb correlates with decreased uptake and cross-presentation of Ab-Ag complexes. In contrast, engagement of FcgammaRIIb is not associated with changes in cross-presentation in mature DCs. We provide evidence that FcgammaRIIb expression is patently reduced in mature DCs, an effect that is modulated by treatment with cytokines. The regulated expression of activating and inhibitory FcgammaRs in DCs emerges as a critical checkpoint in the process of Ag uptake and cross-presentation.     

11 Beta-hydroxysteroid dehydrogenase type 1 is induced in human monocytes upon differentiation to macrophages.

11beta-hydroxysteroid dehydrogenases (11beta-HSD) perform prereceptor metabolism of glucocorticoids through interconversion of the active glucocorticoid, cortisol, with inactive cortisone. Although the immunosuppressive and anti-inflammatory activities of glucocorticoids are well documented, the expression of 11beta-HSD enzymes in immune cells is not well understood. Here we demonstrate that 11beta-HSD1, which converts cortisone to cortisol, is expressed only upon differentiation of human monocytes to macrophages. 11beta-HSD1 expression is concomitant with the emergence of peroxisome proliferator activating receptor gamma, which was used as a surrogate marker of monocyte differentiation. The type 2 enzyme, 11beta-HSD2, which converts cortisol to cortisone, was not detectable in either monocytes or cultured macrophages. Incubation of monocytes with IL-4 or IL-13 induced 11beta-HSD1 activity by up to 10-fold. IFN-gamma, a known functional antagonist of IL-4 and IL-13, suppressed the induction of 11beta-HSD1 by these cytokines. THP-1 cells, a human macrophage-like cell line, expressed 11beta-HSD1 and low levels of 11beta-HSD2. The expression of 11beta-HSD1 in these cells is up-regulated 4-fold by LPS. In summary, we have shown strong expression of 11beta-HSD1 in cultured human macrophages and THP-1 cells. The presence of the enzyme in these cells suggests that it may play a role in regulating the immune function of these cells.     

Individuals co-exposed to sand fly saliva and filarial parasites exhibit altered monocyte function

BackgroundIn Mali, cutaneous leishmaniasis (CL) and filariasis are co-endemic. Previous studies in animal models of infection have shown that sand fly saliva enhance infectivity of Leishmania parasites in naïve hosts while saliva-specific adaptive immune responses may protect against cutaneous and visceral leishmaniasis. In contrast, the human immune response to Phlebotomus duboscqi (Pd) saliva, the principal sand fly vector in Mali, was found to be dichotomously polarized with some individuals having a Th1-dominated response and others having a Th2-biased response. We hypothesized that co-infection with filarial parasites may be an underlying factor that modulates the immune response to Pd saliva in endemic regions.Methodology/Principal findingsTo understand which cell types may be responsible for polarizing human responses to sand fly saliva, we investigated the effect of salivary glands (SG) of Pd on human monocytes. To this end, elutriated monocytes were cultured in vitro, alone, or with SG, microfilariae antigen (MF ag) of Brugia malayi, or LPS, a positive control. The mRNA expression of genes involved in inflammatory or regulatory responses was then measured as were cytokines and chemokines associated with these responses. Monocytes of individuals who were not exposed to sand fly bites (mainly North American controls) significantly upregulated the production of IL-6 and CCL4; cytokines that enhance leishmania parasite establishment, in response to SG from Pd or other vector species. This selective inflammatory response was lost in individuals that were exposed to sand fly bites which was not changed by co-infection with filarial parasites. Furthermore, infection with filarial parasites resulted in upregulation of CCL22, a type-2 associated chemokine, both at the mRNA levels and by its observed effect on the frequency of recruited monocytes.Conclusions/SignificanceTogether, our data suggest that SG or recombinant salivary proteins from Pd alter human monocyte function by upregulating selective inflammatory cytokines.     

Enhancement of human monocyte function against Candida albicans by the colony-stimulating factors (CSF): IL-3, granulocyte-macrophage-CSF, and macrophage-CSF

The effect of IL-3, granulocyte-macrophage (GM)-CSF and macrophage (M)-CSF on Candida albicans growth inhibition by human peripheral blood monocytes was investigated. By using a radiolabel microassay developed in our laboratory that makes use of the incorporation of [3H]glucose into residual C. albicans, we demonstrated that rGM-CSF and rIL-3 effectively enhanced human monocyte-mediated anticandidal activity. Incubation for 24 h with either GM-CSF or IL-3 significantly enhanced monocyte antifungal responses down to 0.01 U/ml. M-CSF, at higher concentrations of 10 U/ml, could also enhance monocyte function but to a smaller degree. None of the CSF interfered directly with fungal growth, even up to 1000 U/ml. Because IFN-gamma is also a known monocyte activator, its effect on monocytes was also assessed. Monocytes were first cultured in medium for several days and then further incubated with each of the cytokines. Monocytes aged in medium were found to lose their spontaneous anticandidal activity. Such aged monocytes did not develop anticandidal activity in response to IFN-gamma but did in response to GM-CSF or IL-3. To further elucidate this difference, fresh monocytes were continuously cultured with or without cytokines for 1 to 5 days before assessing their anticandidal activity. Monocytes cultured in IFN-gamma progressively lost their activity by 2 days but monocytes in GM-CSF or IL-3 maintained their high level of anticandidal activity throughout the whole length of culture. Therefore, GM-CSF and IL-3 not only enhanced fresh monocyte anticandidal activity, but maintained monocyte function for a longer period. These results suggest that GM-CSF and IL-3 may act on monocytes via a different pathway than does IFN-gamma.     

Endoglin modulates cellular responses to TGF-beta 1

Endoglin is a homodimeric membrane glycoprotein which can bind the beta 1 and beta 3 isoforms of transforming growth factor-beta (TGF-beta). We reported previously that endoglin is upregulated during monocyte differentiation. We have now observed that TGF-beta itself can stimulate the expression of endoglin in cultured human monocytes and in the U-937 monocytic line. To study the functional role of endoglin, stable transfectants of U-937 cells were generated which overexpress L- or S- endoglin isoforms, differing in their cytoplasmic domain. Inhibition of cellular proliferation and downregulation of c-myc mRNA which are normally induced by TGF-beta 1 in U-937 cells were totally abrogated in L-endoglin transfectants and much reduced in the S- endoglin transfectants. Inhibition of proliferation by TGF-beta 2 was not altered in the transfectants, in agreement with the isoform specificity of endoglin. Additional responses of U-937 cells to TGF- beta 1, including stimulation of fibronectin synthesis, cellular adhesion, platelet/endothelial cell adhesion molecule 1 (PECAM-1) phosphorylation, and homotypic aggregation were also inhibited in the endoglin transfectants. However, modulation of integrin and PECAM-1 levels and stimulation of mRNA levels for TGF-beta 1 and its receptors R-I, R-II, and betaglycan occurred normally in the endoglin transfectants. No changes in total ligand binding were observed in L- endoglin transfectants relative to mock, while a 1.5-fold increase was seen in S-endoglin transfectants. The degradation rate of the ligand was the same in all transfectants. Elucidating the mechanism by which endoglin modulates several cellular responses to TGF-beta 1 without interfering with ligand binding or degradation should increase our understanding of the complex pathways which mediate the effects of this factor.     

Regulation of myeloid cell function through the CD200 receptor

Myeloid cells play pivotal roles in chronic inflammatory diseases through their broad proinflammatory, destructive, and remodeling capacities. CD200 is widely expressed on a variety of cell types, while the recently identified CD200R is expressed on myeloid cells and T cells. CD200 deletion in vivo results in myeloid cell dysregulation and enhanced susceptibility to autoimmune inflammation, suggesting that the CD200-CD200R interaction is involved in immune suppression. We demonstrate in this study that CD200R agonists suppress mouse and human myeloid cell function in vitro, and also define a dose relationship between receptor expression and cellular inhibition. IFN-gamma- and IL-17-stimulated cytokine secretion from mouse peritoneal macrophages was inhibited by CD200R engagement. Inhibitory effects were not universal, as LPS-stimulated responses were unaffected. Inhibition of U937 cell cytokine production correlated with CD200R expression levels, and inhibition was only observed in low CD200R expressing cells, if the CD200R agonists were further cross-linked. Tetanus toxoid-induced human PBMC IL-5 and IL-13 secretion was inhibited by CD200R agonists. This inhibition was dependent upon cross-linking the CD200R on monocytes, but not on cross-linking the CD200R on CD4+ T cells. In all, we provide direct evidence that the CD200-CD200R interaction controls monocyte/macrophage function in both murine and human systems, further supporting the potential clinical application of CD200R agonists for the treatment of chronic inflammatory diseases.