Transforming growth factor-beta in synovial fluids modulates Fc gamma RII (CD16) expression on mononuclear phagocytes.

Mononuclear phagocytes in the synovium of patients with arthritis, in contrast to blood monocytes, were found to express a third receptor for the constant region of Ig (Fc gamma RIII), in addition to Fc gamma RI and Fc gamma RII. Previously identified on mature mononuclear phagocytes or phagocytes exposed to transforming growth factor-beta (TGF-beta) in vitro, this study documents the presence of Fc gamma RIII (CD16) expressing cells at an inflammatory site. Furthermore, the presence of CD16 on the majority of the LeuM3 (CD14) positive synovial monocytic cells could be mimicked by exposing blood monocytes to synovial fluids from patients with rheumatoid arthritis (17 of 19) and synovial fluids from patients with osteoarthritis (4 of 4). In additional studies, the soluble factor in inflammatory synovial fluids responsible for regulating CD16 expression was found to be consistent with the presence of TGF-beta. Inhibition of the activity in synovial fluids with a neutralizing antibody to TGF-beta confirmed a role for this peptide in synovial phagocytic cell CD16 expression. Moreover, signal transduction through CD16 on synovial phagocytes resulted in augmented extracellular release of superoxide anion that may contribute to tissue damage and other inflammatory sequelae. Identification of TGF-beta and its association with upregulation of CD16 at sites of chronic inflammation may provide insight into the destructive lesions associated with inflammatory arthropathies.     

IL-10 stimulates monocyte Fc gamma R surface expression and cytotoxic activity. Distinct regulation of antibody-dependent cellular cytotoxicity by IFN-gamma, IL-4, and IL-10.

T cell-derived cytokines IFN-gamma and IL-4 have different regulatory effects on two functionally important molecules on human monocytes: MHC class II Ag and the Fc receptor for monomeric IgG, Fc gamma RI (CD64). MHC class II Ag, and Fc gamma RI are both upregulated in the presence of IFN-gamma. IL-4 induces MHC class II Ag expression but reduces Fc gamma RI expression. Recently, we showed that the cytokine IL-10 also affects MHC class II Ag expression. Here, we demonstrate that in contrast to the down-regulation of MHC class II Ag expression, IL-10 stimulates Fc gamma RI expression on human monocytes comparable to the levels of Fc gamma RI expression induced by IFN-gamma. The IL-10-induced Fc gamma RI expression is specific because anti-IL-10 antibodies completely reverse the IL-10-induced surface expression of Fc gamma RI and correlate with an enhanced capacity to lyse anti-D-coated human rhesus-positive erythrocytes. IL-10 fails to induce the expression of Fc gamma RII (CD32) and Fc gamma RIII (CD16). Furthermore, we demonstrate that IL-10 is able to prevent down-regulation in surface membrane expression of all three Fc gamma R that can be found when monocytes are cultured in the presence of IL-4. In contrast to IFN-gamma, IL-10 does not restore the reduced antibody-dependent cellular cytotoxicity (ADCC) activity of IL-4-cultured monocytes. Together, these results show that, similar to IFN-gamma, IL-10 is capable of enhancing Fc gamma R expression and ADCC activity, and that IFN-gamma, IL-4, and IL-10 have different regulatory effects on both monocyte Ag-presenting capacity and ADCC activity.     

IL-4 decreases Fc gamma R membrane expression and Fc gamma R-mediated cytotoxic activity of human monocytes

Monocytes can express three classes of FcR for IgG: Fc gamma RI, Fc gamma RII, and Fc gamma RIII (CD64, CD32, and CD16, respectively) of which the Fc gamma RIII is expressed after prolonged culture. Fc gamma R expression is regulated by IFN-gamma. Because IFN-gamma and IL-4 have antagonistic effects on the expression of the FcR for IgE on human monocytes, we studied the effect of IL-4 on Fc gamma R expression and function. We show that IL-4 down-regulates Fc gamma RI, Fc gamma RII, and Fc gamma RIII expression of cultured monocytes and inhibits IFN-gamma enhanced Fc gamma RI expression. Exposure of monocytes to IL-4 for 40 h resulted in a dose-dependent decrease of the expression of all three Fc gamma R that persisted throughout the whole culture period (7 days). Anti-IL-4 antibodies completely reversed the IL-4 effect. In addition the impaired Fc gamma R expression correlated directly with reduced Fc gamma R-mediated function because monocytes cultured in the presence of IL-4 have a reduced capacity to lyse human E opsonized with human IgG anti-D or mouse antiglycophorin A antibodies. These observations, together with the previous finding that IL-4 induces Fc epsilon RIIb expression on monocytes, indicate that IL-4 and IFN-gamma may control the Fc gamma R-mediated immune response by differentially regulating Fc gamma R expression.     

The significant increase of FcγRIIIA (CD16), a sensitive marker, in patients with coronary heart disease

Our previous studies suggest that Fc receptor III A of immunoglobulin G (FcγRIIIA, also named CD16) is closely correlated to coronary heart disease (CHD). However, whether or not deregulated FcγRIIIA expression is involved in the development of CHD remains largely unclear. Herein, we investigated the FcγRIIIA mRNA expression in the leukocytes, the serum protein level of soluble CD16 (sCD16) and membrane CD16 on monocytes from 100 diagnosed CHD patients and 40 healthy individuals. Our results demonstrated that there was a significant increase of FcγRIIIA at the mRNA level in leukocytes, and at the protein level for both sCD16 in sera and membrane CD16 on monocytes from CHD patients compared to the healthy control. Similarly to the soluble CD14 (sCD14), the level of macrophage colony stimulating factor (M-CSF) in sera was also higher in CHD patients than that in the control individuals. Furthermore, the levels of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1), in sera and the mean fluorescent intensity of intercellular adhesion molecule 1 (ICAM-1, CD54) on CD14(+) CD16(+) monocytes were increased in CHD patients. Overall, these data demonstrated that FcγRIIIA (CD16) is involved in the pathogenesis of CHD by activating monocytes and stimulating inflammation. The significant increase of CD14(+) CD16(+) monocytes in CHD patients therefore suggested that the increase of the FcγRIIIA level might be a sensitive marker for the CHD diagnosis.     

LILRA2 activation inhibits dendritic cell differentiation and antigen presentation to T cells

The differentiation of monocytes into dendritic cells (DC) is a key mechanism by which the innate immune system instructs the adaptive T cell response. In this study, we investigated whether leukocyte Ig-like receptor A2 (LILRA2) regulates DC differentiation by using leprosy as a model. LILRA2 protein expression was increased in the lesions of the progressive, lepromatous form vs the self-limited, tuberculoid form of leprosy. Double immunolabeling revealed LILRA2 expression on CD14+, CD68+ monocytes/macrophages. Activation of LILRA2 on peripheral blood monocytes impaired GM-CSF induced differentiation into immature DC, as evidenced by reduced expression of DC markers (MHC class II, CD1b, CD40, and CD206), but not macrophage markers (CD209 and CD14). Furthermore, LILRA2 activation abrogated Ag presentation to both CD1b- and MHC class II-restricted, Mycobacterium leprae-reactive T cells derived from leprosy patients, while cytokine profiles of LILRA2-activated monocytes demonstrated an increase in TNF-alpha, IL-6, IL-8, IL-12, and IL-10, but little effect on TGF-beta. Therefore, LILRA2 activation, by altering GM-CSF-induced monocyte differentiation into immature DC, provides a mechanism for down-regulating the ability of the innate immune system to activate the adaptive T cell response while promoting an inflammatory response.     

Aberrant expression of FcγRIIIA (CD16) contributes to the development of atherosclerosis

Previous studies have documented that Fc receptor III A of immunoglobulin G (FcγRIIIA, also named CD16) is involved in the development of coronary heart disease (CHD). However, the mechanism responsible for FcγRIIIA's in contribution to CHD development remains largely unclear. Herein, we investigated the possible role of FcγRIIIA in the development of atherosclerosis. Our results showed that the elevated level of FcγRIIIA on monocytes closely correlated to the adhesive efficiency of human umbilical vein endothelial cells (HUVECs) in vitro. Importantly, we also observed increased population of CD16(+) monocytes and elevated CD16 level on monocytes in ApoE(-/-) mice with characterized atherosclerosis after feeding with high-fat diet for 10weeks. The enhancement of CD16 on monocytes closely correlated to increased content of MMP-9 in aorta and increased inflammatory cytokines in sera. In addition, similar to simvastatin, recombinant human M-CSF represented a robust inhibitory influence on plaque instability and inflammation. Taken together, these data established that FcγRIIIA (CD16)-mediated signaling orchestrated by interaction between monocytes and HUVECs, coupled with inflammatory cytokine stimulation and MMP activation, as a fundamental pathway linked to the development of atherosclerotic formation. Inhibition of FcγRIIIA or its signaling thus might represent a promising approach for the prevention and treatment of CHD.     

Regulation of aminopeptidase-N (CD13) and Fc epsilon RIIb (CD23) expression by IL-4 depends on the stage of maturation of monocytes/macrophages.

IL-4 has multiple biologic activities and it has been shown to have effects on B and T lymphocytes, mast cells, NK cells, and monocytes. We studied the influence of IL-4 on the expression of cell membrane determinants, in particular aminopeptidase-N (CD13) and Fc epsilon RIIb (CD23), on human peripheral blood monocytes. We compared the response of monocytes with the response of human alveolar macrophages and monocytic cell lines (U937 and THP1), as mature and more immature representatives of the mononuclear phagocyte system, respectively. A dose-dependent increase of the expression of CD13 Ag was observed when monocytes were cultured with IL-4. Kinetic analyses revealed that this induction was maximal after 2 to 3 days of culture and resembled the kinetics of IL-4-induced expression of Fc epsilon RIIb on monocytes. This IL-4-induced increase was absent when monocytes were cultured with IL-4 and an anti-IL-4 antiserum. Concomitantly, an IL-4-induced increase in leucine-aminopeptidase activity could be observed. Northern blot analysis showed that incubation of monocytes with IL-4 induced a marked increase in CD13 mRNA. Alveolar macrophages also exhibited an increase in CD13 Ag expression when exposed to IL-4. Surprisingly, IL-4 was unable to induce expression of Fc epsilon RIIb on alveolar macrophages. U937 and THP1 cells did not show an induction of CD13 Ag when cultured in the presence of IL-4. However, IL-4 did induce the expression of Fc epsilon RIIb on both cell lines, suggesting the presence of functional IL-4R. Our data demonstrate that IL-4 increases the expression of CD13 Ag on monocytes. This IL-4-induced increase can also be observed in more mature monocytic cells such as alveolar macrophages, but is absent in immature cells such as U937 or THP1 cells. This is functionally accompanied by an increase in leucine-aminopeptidase activity and may be part of the general activation of monocytes/macrophages by IL-4. In conclusion, the data suggest that IL-4 responsiveness, in particular the induction of CD13 Ag and Fc epsilon RIIb expression, may be dependent on the stage of maturation of monocytes/macrophages.     

Elastin receptor (spliced galactosidase) occupancy by elastin peptides counteracts proinflammatory cytokine expression in lipopolysaccharide-stimulated human monocytes through NF-kappaB down-regulation

In inflammatory diseases, strong release of elastinolytic proteases results in elastin fiber degradation generating elastin peptides (EPs). Chemotactic activity for inflammatory cells was, among wide range of properties, the former identified biological activity exerted by EPs. Recently, we demonstrated the ability of EPs to favor a Th1 cytokine (IL-2, IFN-gamma) cell response in lymphocytes and to regulate IL-1beta expression in melanoma cells. We hypothesized that EPs might also influence inflammatory cell properties by regulating cytokine expression by these cells. Therefore, we investigated the influence of EPs on inflammatory cytokine synthesis by human monocytes. We evidenced that EPs down-regulated both at the mRNA and protein levels the proinflammatory TNF-alpha, IL-1beta, and IL-6 expression in LPS-activated monocytes. Such negative feedback loop could be accounted solely for EP-mediated effects on proinflammatory cytokine production because EPs did not affect anti-inflammatory IL-10 or TGF-beta secretion by LPS-activated monocytes. Furthermore, we demonstrated that EP effect on proinflammatory cytokine expression by LPS-stimulated monocytes could not be due either to a decrease of LPS receptor expression or to an alteration of LPS binding to its receptor. The inhibitory effects of EPs on cytokine expression were found to be mediated by receptor (spliced galactosidase) occupancy, as being suppressed by lactose, and to be associated with the decrease of NF-kappaB-DNA complex formation. As a whole, these results demonstrated that EP/spliced galactosidase interaction on human monocytes down-regulated NF-kappaB-dependent proinflammatory cytokine expression and pointed out the critical role of EPs in the regulation of inflammatory response.     

TGF-beta 1 prevents the noncognate maturation of human dendritic Langerhans cells

TGF-beta 1 is critical for differentiation of epithelial-associated dendritic Langerhans cells (LC). In accordance with the characteristics of in vivo LC, we show that LC obtained from human monocytes in vitro in the presence of TGF-beta 1 1) express almost exclusively intracellular class II Ags, low CD80, and no CD83 and CD86 Ags and 2) down-regulate TNF-RI (p55) and do not produce IL-10 after stimulation, in contrast to dermal dendritic cells and monocyte-derived dendritic cells. Surprisingly, while LC exhibit E-cadherin down-regulation upon exposure to TNF-alpha and IL-1, TGF-beta 1 prevents the final LC maturation in response to TNF-alpha, IL-1, and LPS with respect to Class II CD80, CD86, and CD83 Ag expression, loss of FITC-dextran uptake, production of IL-12, and Ag presentation. In sharp contrast, CD40 ligand cognate signal induces full maturation of LC and is not inhibited by TGF-beta 1. The presence of emigrated immature LCs in human reactive skin-draining lymph nodes provides in vivo evidence that LC migration and final maturation may be differentially regulated. Therefore, due to the effects of TGF-beta 1, inflammatory stimuli may not be sufficient to induce full maturation of LC, thus avoiding potentially harmful immune responses. We conclude that TGF-beta 1 appears to be responsible for both the acquisition of LC phenotype, cytokine production pattern, and prevention of noncognate maturation.     

CD16 regulates TRIF-dependent TLR4 response in human monocytes and their subsets

Blood monocytes recognize Gram-negative bacteria through the TLR4, which signal via MyD88- and TRIF-dependent pathway to trigger an immune-inflammatory response. However, a dysregulated inflammatory response by these cells often leads to severe pathologies such as sepsis. We investigated the role of CD16 in the regulation of human monocyte response to Gram-negative endotoxin and sepsis. Blood monocytes from sepsis patients demonstrated an upregulation of several TRIF-dependent genes as well as a selective expansion of CD16-expressing (CD16(+)) monocytes. Gene expression and biochemical studies revealed CD16 to regulate the TRIF-dependent TLR4 pathway in monocytes by activating Syk, IFN regulatory factor 3, and STAT1, which resulted in enhanced expression of IFNB, CCL5, and CXCL10. CD16 also upregulated the expression of IL-1R-associated kinase M and IL-1 receptor antagonist, which are negative regulators of the MyD88-dependent pathway. CD16 overexpression or small interfering RNA knockdown in monocytes confirmed the above findings. Interestingly, these results were mirrored in the CD16(+) monocyte subset isolated from sepsis patients, providing an in vivo confirmation to our findings. Collectively, the results from the current study demonstrate CD16 as a key regulator of the TRIF-dependent TLR4 pathway in human monocytes and their CD16-expressing subset, with implications in sepsis.     

Lipopolysaccharide and interleukin 1 inhibit interferon-gamma-induced Fc receptor expression on human monocytes

The objectives of these studies were to study the effects of bacterial lipopolysaccharide (LPS) on interferon-gamma (IFN-gamma)-induced Fc receptor expression on human monocytes and to examine whether these effects were mediated through stimulation of interleukin 1 (IL-1) production. Fc receptor expression was determined by binding of monomeric monoclonal murine immunoglobulin (Ig)G2a and cytofluorographic analysis. IL-1 activity in monocyte supernatants and lysates was assayed by augmentation of mitogen-induced murine thymocyte proliferation. IFN-gamma induced the expression of Fc receptors on human monocytes that were specific for murine IgG2a. This induction was inhibited by the addition of LPS in amounts as low as 2 to 8 pg/ml. LPS inhibition of IFN-gamma-induced Fc receptor expression was paralleled by the appearance of IL-1 in monocyte lysates and supernatants. The addition of purified human or recombinant IL-1 beta at the initiation of culture similarly inhibited the expression of IFN-gamma-induced Fc receptors on the monocytes. LPS also inhibited Fc receptor expression on the human myelomonocytic cell line THP-1 after induction with IFN-gamma or phorbol myristate acetate alone or with both agents together. This inhibition also was paralleled by the production of IL-1 but the addition of exogenous IL-1 to the THP-1 cells had no effect on IFN-gamma-induced Fc receptor expression. Tumor necrosis factor (TNF) inhibited IFN-gamma-induced Fc receptor expression on human monocytes but was much less potent than comparable amounts of IL-1. TNF also did not inhibit Fc receptor expression on THP-1 cells. In fact, IL-1 or TNF led to an enhancement in IFN-gamma-induced Fc receptor expression on THP-1 cells. These results indicate that LPS can inhibit IFN-gamma-induced Fc receptor expression on human monocytes and that IL-1 and TNF may mediate these effects of LPS. Thus, an autocrine or paracrine role is suggested for these cytokines. The possibility exists that intracellular IL-1 resulting from LPS stimulation may be at least in part responsible for inhibition of Fc receptor expression.     

Transforming growth factor-beta is a potent immunosuppressive agent that inhibits IL-1-dependent lymphocyte proliferation

Transforming growth factor-beta (TGF-beta), a product of neoplastic and hemopoietic cells, is a bifunctional regulator of the immune response. At femtomolar concentrations, TGF-beta stimulates monocyte migration, and picomolar quantities induce synthesis of monocyte growth factors, including IL-1, that may promote tissue repair by regulating fibrosis and angiogenesis. Paradoxically, TGF-beta at picomolar concentrations also blocks the ability of IL-1 to stimulate lymphocyte proliferation. At 0.01 to 1.0 ng/ml, TGF-beta 1 and its homologue, TGF-beta 2, suppress the IL-1-dependent murine thymocyte proliferation assay. TGF-beta also inhibits human peripheral blood T lymphocyte mitogenesis. Inhibition of cell division appears to occur after activation of the lymphocytes inasmuch as neither gene expression nor translation of IL-2R is suppressed. Furthermore, TGF-beta does not block synthesis of IL-2. Therefore, TGF-beta 1 and TGF-beta 2 likely act at a site distal to IL-1 to block lymphocyte DNA synthesis. These findings suggest that TGF-beta secreted in an inflammatory site may be beneficial in diminishing lymphocyte function while promoting fibrosis and tissue repair. However, TGF-beta generated by neoplastic tissues may provide a mechanism for unrestricted tumor cell growth through its selective immunosuppressive effects.     

Regulation of TGF-beta response during T cell activation is modulated by IL-10.

TGF-beta1 is an important pleiotropic cytokine that has been described to have both stimulatory and inhibitory effects on cell growth and differentiation. For several cell types, the effect of TGF-beta1 was found to correlate with the differentiation stage of the cells and the presence of other cytokines. In this report, we address the influence of TGF-beta1 on CD4(+) T cell activation by evaluating the effect of TGF-beta1 on the proliferative and cytokine responses of purified resting and activated human or mouse CD4(+) T cells. TGF-beta1 inhibits proliferation and cytokine secretion on resting CD4(+) T cells but has no inhibitory effect on activated T cells. Moreover, TGF-beta1 unresponsiveness of activated T cells was correlated with a down-regulation in the expression of the TGF-beta receptor type II. Interestingly, IL-10 addition enhances TGF-beta receptor type II expression and restores TGF-beta responsiveness on activated T cells. These results indicated that TGF-beta responsiveness is sequentially regulated on T cells by the modulation of the of TGF-beta receptor type II chain expression. Moreover, we have identified a novel regulatory role of IL-10 on TGF-beta-dependent T cell growth that can explain the control of T cell activation on chronic vs acute inflammatory sites.     

Differential modulation of stimulatory and inhibitory Fc gamma receptors on human monocytes by Th1 and Th2 cytokines

Immune complex-mediated inflammatory responses are initiated by Fc gamma R on phagocytes. We report in this study that an inhibitory receptor, Fc gamma RIIb2, is expressed on circulating human monocytes, and when co-cross-linked with stimulatory Fc gamma R it down-regulates effector function. Fc gamma RIIb2 expression is increased by IL-4 and decreased by IFN-gamma, in contrast to the activating receptor, Fc gamma RIIa, which is increased by IFN-gamma and decreased by IL-4. Thus, Th1 and Th2 cytokines differentially regulate the opposing Fc gamma R systems, altering the balance of activating and inhibiting Fc gamma R. The detection and cytokine modulation of Fc gamma RIIb2 in human myeloid cells provide evidence of a negative regulator of immune complex-mediated responses in human phagocytes and offer a new approach to limit Ab-triggered inflammation in autoimmune disease.     

Immune dysfunction in mice with plasmacytomas. I. Evidence that transforming growth factor-beta contributes to the altered expression of activation receptors on host B lymphocytes.

Plasmacytoma-bearing mice (PC-mice) develop a polyclonal B cell immunodeficiency syndrome characterized by marked impairment of: a) primary antibody responses and b) proliferative responses to B cell mitogens. The present investigations used two-color flow cytometry to examine B lymphocytes from the spleens and lymph nodes of PC-mice and found decreased surface membrane expression of surface IgM (sIgM), transferrin receptors (TfR) and IgE FcR (CD23), increased expression of class II MHC, but normal expression of B220, Mel-14, Fc gamma RII, and Fc mu R. These changes were not related to the H chain class or the amount of Ig produced by the plasmacytoma. When cultured with IL-4, B lymphocytes from PC-mice increased their expression of sIgM and class II MHC, but not of CD23. Several findings implicate transforming growth factor-beta 1 (TGF-beta 1) in the mechanism that modulates receptor expression on B lymphocytes in PC-mice: a) ascites fluid from PC-mice contains large quantities of TGF-beta 1; b) supernatants of cultured spleen cells from PC mice contain up to eightfold more TGF-beta than is found with normal spleen cells; c) cloned plasmacytoma cells produce TGF-beta in vitro; and d) the abnormal phenotype of B cells from PC-mice, i.e., decreased CD23, sIgM, and TfR, and increased class II MHC, is induced on normal B cells cultured in the presence of TGF-beta 1. Because sIgM, TfR, class II MHC, and CD23 are molecules that play fundamental roles in the activation of normal B cells, their modulation by TGF-beta 1: a) identifies molecular mechanisms that could account for some of the known immunosuppressive properties of TGF-beta 1 and b) implicates TGF-beta in the pathogenesis of the polyclonal B cell immunodeficiency that is characteristic of plasma cell tumors.     

Monocytes stimulated by 110-kDa fibronectin fragments suppress proliferation of anti-CD3-activated T cells

One hundred ten to 120-kDa fragments of fibronectin (FNf), generated by proteases released in the course of tissue injury and inflammation, stimulate monocytes to produce proinflammatory cytokines, promote mononuclear leukocytes (MNL) transendothelial migration, up-regulate monocyte CD11b and CD86 expression, and induce monocyte-derived dendritic cell differentiation. To investigate whether the proinflammatory consequences of FNf are offset by responses that can suppress proliferation of activated T lymphocytes, we investigated the effect of FNf-treated MNL on autologous T lymphocytes induced to proliferate by substrate-immobilized anti-CD3. FNf-stimulated MNL suppressed anti-CD3-induced T cell proliferation through both contact-dependent and contact-independent mechanisms. Contact-independent suppression was mediated, at least in part, by IL-10 and TGF-beta released by the FNf-stimulated MNL. After 24-48 h exposure to FNf, activated T cells and monocytes formed clusters displaying CD25, CD14, CD3, and CD4 that were not dissociable by chelation of divalent cations. Killing monocytes with l-leucine methyl ester abolished these T cell-monocyte clusters and the ability of the FNf-stimulated MNL to suppress anti-CD3 induced T cell proliferation. Thus, in addition to activating MNL and causing them to migrate to sites of injury, FNf appears to induce suppressor monocytes.     

Sulfonated human immunoglobulin enhances CD16-linked CD11b expression on human neutrophils

Intravenous human immunoglobulin therapy infrequently results in excessive inflammatory responses in vivo; these effects are not fully understood. We assessed whether sulfonated human immunoglobulin (SHIG) or polyethylene glycol-treated human immunoglobulin (PHIG) enhanced expression of inflammatory receptors on peripheral blood neutrophils in vitro, such as alphaMbeta2 (CD11b/CD18) and Fc gamma receptor type III (FcgammaRIII). CD11b and CD16 expression on neutrophils was measured by fluorescence flow cytometry. Various cytokines were assessed using a highly sensitive fluorescence microsphere system. SHIG enhanced/induced CD11b expression and partial aggregations on neutrophils, but PHIG did not. No detection of aggregation IgG was observed in SHIG and PHIG. SHIG-induced CD11b expression was inhibited by treatment of corticosteroid (dexamethasone) and by anti-CD16 monoclonal antibody. Concentrations of various cytokines such as interleukin (IL)-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, RANTES, tumor necrosis factor (TNF)-alpha, and interferon (INF)-gamma in culture supernatant were not significantly changed by SHIG or PHIG. SHIG and PHIG did not enhance CD16 on neutrophils. SHIG enhanced CD16-linked CD11b expression on neutrophils in vitro. CD11b induction was inhibited by dexamethasone and by anti-CD16 antibody. These in vitro results suggest that aggregations and enhancement of CD11b on neutrophils by SHIG may induce excessive inflammatory responses in vivo.     

Important roles of CD32 in promoting suppression of IL-4 induced immune responses by a novel anti-IL-4Rα therapeutic antibody

ABSTRACT

Asthma is characterized by airway hyperresponsiveness and inflammation, as well as underlying structural changes to the airways. Interleukin-4 (IL-4) is a key T-helper type 2 (Th2) cytokine that plays important roles in the pathogenesis of atopic and eosinophilic asthma. We developed a novel humanized anti-IL-4Rα antibody that can potently inhibit IL-4/IL-13-mediated TF-1 cell proliferation. Using monocytes isolated from human peripheral blood mononuclear cells (PBMCs), we revealed a critical role of CD32 in modulating the immune responses of monocytes in response to blockade of IL-4Rα signaling pathway. We, therefore, devised a new strategy to increase the efficacy of the anti-IL-4Rα monoclonal antibody for the treatment of asthma and other atopic diseases by co-engaging CD32 and IL-4Rα on monocytic cells by choosing IgG classes or Fc mutations with higher affinities for CD32. The antibody with selectively enhanced affinity for CD32A displayed superior suppression of IL-4-induced monocytes’ activities, including the down-regulation of CD23 expression. Intriguingly, further analysis demonstrated that both CD32A and CD32B contributed to the enhancement of antibody-mediated suppression of CD23 expression from monocytes in response to blockade of IL-4Rα signaling. Furthermore, inhibition of IgE secretion from human PBMC by the antibody variants further suggests that the complex allergic inflammation mediated by IL-4/IL-4Rα signaling might result from a global network where multiple cell types that express multiple FcγRs are all involved, of which CD32, especially CD32A, is a key mediator. In this respect, our study provides new insights into designing therapeutic antibodies for targeting Th2 cytokine-mediated allergic pathogenesis.