Study on the expression of HLA-DR antigens, Fc IgG receptors and C3 complement component on lung macrophages in the primary lung carcinoma

Expression of HLA-DR antigens and Fc IgG receptors as well as C3 complement component, was studied on patients with the primary lung carcinoma and in the control group of nonsmokers and smokers. Macrophages were isolated by broncho-alveolar lavage using bronchofiberoscope. The study was carried out before the treatment, within its course and during remission. The percentage of macrophages with HLA-DR antigens and Fc IgG receptors and C3 complement was observed to diminish before the treatment, after chemo- and radiotherapy as compared to the control. In the course of alpha interferon-treatment and at remission, the expression of HLA-DR antigens on macrophages increased in relation to the remaining groups of patients (p less than or equal to 0.05). The expression of Fc IgG receptors and C3 complement revealed no statistically significant differences. As the findings show the expression of HLA-DR antigens is modulated by interferon and has a tendency to increase in the course of convalescence. On the other hand, the expression of RFc IgG and RC3 complement shows great stabilization in the primary lung carcinoma and undergoes no changes either in the course of treatment or remission.     

HIV-1 infection of monocyte-derived macrophages reduces Fc and complement receptor expression.

Fc receptor (FcR) and complement receptor (CR) expression on HIV-infected monocyte-derived macrophages may be an important determinant of immune function. We studied the effects of HIV-1 infection of macrophages in vitro on FcR and CR expression. Macrophages were infected with HIV-1DV 7 days following isolation, and the expression of Fc gamma RI-III and CR3 were measured at intervals thereafter by flow cytometry. We found a reduction in receptor expression with the percentage of cells expressing FcRI 14 days post infection declining from 77% to 13%, FcRII fell from 96% to 85%, FcRIII from 45% to 9%, and CR3 from 91% to 67% 14 days following infection. As these receptors are important for macrophage function, their down-modulation may contribute to the pathogenesis of HIV-related disease.     

Inhibition of human monocyte Fc receptor and HLA-DR antigen expression by pregnancy alpha-2 glycoprotein.

The precise factor responsible for the altered immunological reactivity and diminished inflammatory responses occurring in pregnancy is unknown. Recent observations implicate the pregnancy-associated alpha-2 glycoprotein (PAG). Using rosette assays for the detection of cells carrying Fc receptors and for the demonstration of surface HLA-DR antigens, we demonstrate that a serum protein fraction rich in PAG inhibits these surface markers on human monocytes. Brief exposure of normal peripheral blood monocytes to physiological concentrations of PAG led to almost total loss of Fc receptor expression, an effect that was concentration-related. Using a monoclonal anti-HLA-DR antibody linked to red cells, physiological concentrations of PAG also significantly inhibited the detection of HLA-DR on monocytes. As controls, we used a protein fraction isolated in identical fashion from male sera devoid of PAG, and an alpha-2 macroglobulin isolate. These findings suggest that PAG, by masking monocyte surface markers, could be responsible for the suppression of some cell functions during pregnancy.     

Isolation of various canine leucocytes and their characterization by surface marker analysis.

Various techniques were used to separate canine peripheral blood leucocytes into populations enriched in lymphocytes, polymorphonuclear leucocytes, phagocytic mononuclear cells (monocytes) and macrophages. Surface markers on each cell population were determined by rosette formation. Fc receptors for IgG and complement receptors (C3b and C3d) were present on PMN, monocytes, macrophages as well as on a sub-population of lymphocytes. Purification of the lymphocytes into T-and B-cell-enriched populations revealed that these receptors were present only on the B lymphocytes and not on the T lymphocytes. In addition, a third lymphocyte population, which did not possess surface immunoglobulin, and Fc receptor but not the complement receptor. None of the cell populations exhibited C4 complement receptors or Fc receptors for IgM. When different cell populations were tested for their ability to form rosettes directly with human type 'O' red blood cells it was found that most populations could rosette, suggesting that this technique could not be used as a specific marker for canine T lymphocytes.     

Modulation of Fc and C3b receptor expression on guinea-pig macrophages by lymphokines.

The decrease in Fc-receptor-positive cells that occurred during a 6 h incubation of resident and elicited guinea-pig macrophages was partly abrogated when lymphokines were present in the culture. When the same lymphokine preparations were tested on C3b receptor-expression they preferentially sustained the percentage of C3b rosettes formed by resident rather than elicited macrophages. This lymphokine-induced maintenance of Fc and C3b rosettes by cultured macrophages may have been due to an inhibition of receptor release or an increase in receptor synthesis. Supernatants from cultured macrophages contain shed Fc and C3b receptors which inhibit rosette formation by other macrophages. From the demonstration that culture supernatants from both lymphokine-treated and untreated macrophages significantly inhibited Fc and C3b rosette formation by freshly obtained macrophages it seems that the shedding of Fc and C3b receptors is not modified by lymphokines. The maintenance of Fc and C3b rosettes by lymphokines was inhibited by treatment of the macrophages with cycloheximide, suggesting that the lymphokine effect was due to an increase in synthesis de novo of the Fc and C3b receptors. The lymphokine-inducing antigens, BGG and PPD, and control lymphokine preparations were devoid of receptor modifying activity. The reduction in the percentage of Fc rosettes after 6 h culture appears to be due to a loss of Fc receptors for IgG1. Although lymphokines partly inhibited this effect they could not prevent the loss of these receptors following 24 h culture, unlike their action in augmenting the expression of Fc receptors for IgG2. These findings suggest that a selective enhancement of Fc receptor synthesis by lymphokines may modify the functional activities of macrophages.     

Fcγ receptor expression on splenic macrophages in adult immune thrombocytopenia

Splenic macrophages play a key role in immune thrombocytopenia (ITP) pathogenesis by clearing opsonized platelets. Fcγ receptors (FcγR) participate in this phenomenon, but their expression on splenic macrophages and their modulation by treatment have scarcely been studied in human ITP. We aimed to compare the phenotype and function of splenic macrophages between six controls and 24 ITP patients and between ITP patients according to the treatments they received prior to splenectomy. CD86, human leucocyte antigen D‐related (HLA‐DR) and FcγR expression were measured by flow cytometry on splenic macrophages. The major FcγR polymorphisms were determined and splenic macrophage function was assessed by a phagocytosis assay. The expression of the activation markers CD86 and HLA‐DR was higher on splenic macrophages during ITP compared to controls. While the expression of FcγR was not different between ITP and controls, the phagocytic function of splenic macrophages was reduced in ITP patients treated with intravenous immunoglobulin (IVIg) within the 2 weeks prior to splenectomy. The FCGR3A (158V/F) polymorphism, known to increase the affinity of FcγRIII to IgG, was over‐represented in ITP patients. Thus, these are the first results arguing for the fact that the therapeutic use of IVIg during human chronic ITP does not modulate FcγR expression on splenic macrophages but decreases their phagocytic capabilities.     

Phagocytic cells internalize ZnO particles by FcγII/III-receptor pathway

The present study investigates the process of internalization for bulk ZnO particles in macrophages, and further elucidates the underlying mechanism. Since macrophages are active phagocytes and phagocytosis is a size dependent phenomenon, therefore we hypothesized that bulk ZnO may internalize into macrophages by phagocytic pathways. Interestingly, the phagocytic activity got enhanced in bulk ZnO treated macrophages. Moreover, the bulk ZnO treated macrophages internalized via FcγR-II/III, complement and scavenger–receptor pathways. To confirm the specificity of phagocytic pathway, the uptake was also analyzed in splenocytes where phagocytic (monocytes) and non-phagocytic cells (lymphocytes) are present. It was observed that no significant uptake of bulk ZnO in case of lymphocytes whereas significant uptake in monocytes. Henceforth, our quest for uptake mechanisms also revealed that severe plasma membrane extensions (pseudopodia), FcγR clustering over the surface of macrophages and activation of FcγR signaling were the key players for bulk ZnO uptake; whereas clathrin or caveolae mediated endocytic pathways contributed less. Uptake of these particles was further strengthened by the ZnO-induced activation of the Src-kinase p-Lyn, phospho-tyrosine kinases Syk (spleen tyrosine kinase), p-PLC-γ and PI3K (phosphatidylinositol 3-kinase). Our findings illustrate that the phagocytic nature of macrophages could have led to higher uptake of bulk ZnO.     

Enhanced monocyte Fc phagocytosis by a homologue of interleukin-10 encoded by human cytomegalovirus

Human cytomegalovirus (HCMV) expresses several homologues of human interleukin 10 (hIL-10) possessing immunomodulatory properties which may promote viral infection by modulating the function of myeloid cells. We examined the phenotype and phagocytic capability of human monocytes exposed to hIL-10, an HCMV-encoded hIL-10 homologue expressed during the productive phase of infection (cmvIL-10), and a differentially spliced form of cmvIL-10 expressed during latent and productive phases of infection, (LAcmvIL-10). hIL-10 and cmvIL-10 upregulated expression of Fcγ receptors, stimulated phagocytosis of IgG-opsonised erythrocytes and decreased MHC class II (HLA-DR) expression on purified monocytes within 24 h. In contrast, LAcmvIL-10 decreased HLA-DR expression at later times (48 h and 72 h) but did not increase Fcγ receptor expression. We conclude that cmvIL-10 promotes differentiation of monocytes towards a pro-phagocytic phenotype and that LAcmvIL-10 does not affect monocytes by the same mechanism as cmvIL-10. The significance of these properties to cytomegalovirus pathogenesis is discussed.     

IL-10 augments CD23 expression on U937 cells and down-regulates IL-4-driven CD23 expression on cultured human blood monocytes: effects of IL-10 and other cytokines on cell phenotype and phagocytosis.

The effects of human recombinant interleukin-10 (IL-14) on the expression of several markers on U937 and human peripheral blood monocytes was studied by immunofluorescence and fluorescence-activated cell sorter (FACS) analysis. IL-10 augmented Fc IgE receptor (Fc epsilon RII/CD23) further enhanced by cotreatment with IL-4 or interferon-gamma (IFN-gamma). In contrast, the basal level of Fc epsilon RII expression on blood monocytes appeared to fall in response to IL-10, and this effect became more evident on IL-4-treated cells. Furthermore, the constitutive and IFN-gamma-triggered Fc gamma RI/CD64 expression was augmented on both monocytes and U937 cells. Thus the expression of Fc gamma RII/CD32, Fc gamma/RIII/CD16, Fc alpha R/CD89, the receptor for complement components (CR1/CD35, CD3/CD11b, CR4/CD11c) and the receptor for transferrin/CD71 was not significantly influenced on IL-10-treated cells. IL-10 modestly triggered CD14 antigen expression on monocytes but not U937. The expression of intercellular adhesion molecule-1 (ICAM-1)/CD54 on monocytes was significantly inhibited by IL-10. As expected, a marked reduction of the constitutive as well as of the IFN-gamma or IL-4-driven expression on HLA-DR, HLA-DP and HLA-DQ was observed on IL-10-cultured monocytes. On the other hand, the expression of major histocompatibility complex (MHC) class I molecules was slightly and dose-dependently induced on IL-10-treated monocytes. The ability of blood monocytes to phagocytose IgG-sensitized ox erythrocytes, and to bind and ingest opsonized Escherichia coli or latex particles, was amplified by IL-10. Our data demonstrate that IL-10 modulates the expression of a wide variety of structures on human mononuclear phagocytes, and augments their phagocytic capacity.     

Decreased Fc receptor expression on innate immune cells is associated with impaired antibody-mediated cellular phagocytic activity in chronically HIV-1 infected individuals

In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection.     

TLR7/9-mediated monocytosis and maturation of Gr-1(hi) inflammatory monocytes towards Gr-1(lo) resting monocytes implicated in murine lupus

Circulating monocytes are divided into two major, phenotypically and functionally distinct subsets: Gr-1^hi “inflammatory” and Gr-1^lo “resting” monocytes. One of the unique cellular abnormalities in lupus-prone mice is monocytosis, which is characterized by a selective expansion of Gr-1^lo monocytes and dependent on the expression of stimulatory IgG Fc receptors (FcγR). We speculated that IgG immune complexes containing nuclear antigens could stimulate Gr-1^hi monocytes through interaction with FcγRs and then TLR7 and TLR9, thereby promoting the maturation towards Gr-1^lo monocytes. In the present study, we assessed this hypothesis by analyzing effects of TLR9 or TLR7 agonist on monocytes in vivo. The analysis of various surface markers differentially expressed on both subsets of monocytes in combination with selective depletion of either subset revealed that within 48 h after injection of the TLR9 agonist CpG, approximately one third of Gr-1^hi monocytes became phenotypically identical to Gr-1^lo monocytes. In addition, we observed approximately two-fold increases in the total monocyte population 8-24 h after injection of CpG. Moreover, the activation of TLR9 resulted in an increased expression of stimulatory FcγRIV relative to inhibitory FcγRIIB on monocytes, thereby enhancing their responsiveness to IgG immune complexes. Essentially identical results were obtained after stimulation of TLR7 with a synthetic agonist (1V136). Our results indicate that the activation of TLR7 and TLR9 not only induced the maturation of a fraction of Gr-1^hi monocytes towards Gr-1^lo monocytes but also promoted the overall generation of monocytes, thereby supporting the critical role of TLR7 and TLR9 for the development of monocytosis in lupus-prone mice.     

Colony-stimulating factors and interferon-gamma differentially affect cell surface molecules shared by monocytes and neutrophils

Monocytes and neutrophils share a common progenitor and perform many similar functions, as reflected in the expression of several shared membrane molecules. We show here that such molecules can be independently regulated by the cytokines interferon-gamma (IFN-gamma) and the colony-stimulating factors (CSF) in the context of the cell type on which they are present. Thus, IFN-gamma causes neutrophils to express the high-affinity receptor for IgG, Fc gamma RI, which has been considered to be a monocyte-specific receptor. Although neutrophil Fc gamma RI never reaches the levels present on monocytes, it is induced more rapidly and by lower amounts of IFN-gamma than monocyte Fc gamma RI. Of the CSF, only macrophage (M) CSF has an effect which is to cause a decrease in expression of monocyte Fc gamma RI. Secondly, after culture monocytes express Fc gamma RIII which is constitutively expressed by neutrophils. Although neutrophil Fc gamma RIII can be readily modulated by IFN-gamma, granulocyte (G) CSF and granulocyte-macrophage (GM) CSF, these cytokines do not alter the low levels of monocyte Fc gamma RIII. Thirdly, expression of the gp55 protein recognized by CD14 monoclonal antibodies is decreased after exposure of monocytes to IFN-gamma. Neutrophils express low levels of CD14 and, in this case, IFN-gamma causes an increase in the CD14 antigen on these cells. Of all the molecules investigated, only HLA class II is confined to monocytes, with increases in expression induced by IFN-gamma but not by the CSF.     

Role of monocytes in anti-CD3-induced T-cell DNA synthesis: Effect of chloroquine and monensin on anti-CD3-induced human T-cell activation

Anti-CD3 monoclonal antibody (MoAb) induces proliferation of freshly isolated peripheral blood T cells only in the presence of monocytes/macrophages and requires binding of the Fc portion of antibody to monocytes/macrophages. In this investigation, we examined whether monocytes process anti-CD3 similar to any soluble antigen and present to T cells in context with HLA-DR to induce maximal DNA synthesis. Adherent monocytes were pulsed with anti-CD3 MoAb in the presence or absence of the lysozomotropic agents chloroquine and monensin, which are known to inhibit processing of soluble antigens, washed extensively, and then incubated with autologous T cells in the absence of soluble anti-CD3, and³H-thymidine incorporation and CD25 expression were measured. Both monensin and chloroquine inhibited anti-CD3-pulsed monocyte-induced T-cell DNA synthesis and CD25 expression in a dose-dependent manner. This inhibitory effect was not due to any loss in cell viability or the effect on the expression of HLA-DR on monocytes. Paraformaldehyde-fixed monocytes pulsed with anti-CD3 MoAb induced significantly less DNA synthesis, HLA-DR expression, and CD25 antigen expression on autologous T cells as compared to responses induced by unfixed anti-CD3-pulsed monocytes. The treatment of anti-CD3-pulsed monocytes with frame-work-specific anti-HLA-DR MoAb inhibited their capacity to induce T-cell DNA synthesis. These data suggest that monocytes, in addition to serving as the matrix for cross-linking, also process anti-CD3 MoAb and present to the T cells in the context of HLA-DR antigens to induce optimal DNA synthesis.     

Receptor associated defects of cultured monocytes in bronchial carcinoma

Using peripheral blood monocytes from individuals with bronchial carcinoma (BC) we have measured changes, over an 8 day culture period, in monocyte/macrophage complement (C3b) and IgG (Fc) rosettes, chemotactic factor (CF)-induced enhancement of C3b and IgG (Fc) rosettes, and the phagocytic index (PI) for yeast particles. The same measurements were made on monocyte/macrophages from individuals with non-malignant respiratory diseases (NMRD) and healthy controls (HC). Following 8 days of culture there was a significant increase in C3b rosettes, IgG (Fc) rosettes and the PI in NMRD and HC. In contrast there was no significant increase in C3b rosettes in BC. The PI increased in BC, but on day 8 was still significantly less than control monocytes/macrophages. IgG (Fc) rosettes increased in BC following culture to a similar degree as that observed with NMRD and HC. In addition, there were no differences between BC and HC in CF-induced enhancement of IgG (Fc) rosettes at either day 0 or day 8 of culture whereas complement receptor enhancement in BC was impaired both at the beginning and end of the 8 day culture period. These results indicate that in advanced BC there is an abnormality in the expression of monocyte/macrophage complement receptors (which is not readily demonstrable for IgG [Fc]receptors) and that the defect persists following an 8 day culture period.     

Impaired dendritic cell differentiation and maturation in the absence of C3

Human monocytes can be differentiated into immature dendritic cells (DCs) in the presence of serum and cytokines. One of the main functions of immature DCs is to capture and process antigens. Following maturation, they differentiate into antigen presenting cells. The role of complement in the differentiation process from monocytes towards immature DCs remains elusive. Here we demonstrate that complement 3 (C3) has a regulatory impact on the expression of specific DC surface molecules and DC-derived cytokine production during DC differentiation. We isolated human adherent peripheral blood mononuclear cells, which were cultured in the presence of GM-CSF plus IL-4 in medium supplemented with normal human serum or C3 deficient serum. The lack of C3 during DC differentiation negatively impacted the expression of C-type lectin receptor DC-SIGN, the antigen presenting molecules HLA-DR and CD1a, and the costimulatory molecules CD80 and CD86. Further, the spontaneous production of IL-6 and IL-12 was reduced in the absence of C3. Moreover, the maturation of immature DCs in response to LPS challenge was impaired in the absence of C3 as evidenced by reduced MHC-II, co-stimulatory molecule expression as well as modulated IL-12 and TNF-alpha production. Collectively, our results provide evidence for a novel role of C3 as a critical cofactor in human DC differentiation and maturation.     

In vitro and in vivo macrophage function can occur independently of SLP-76

Expression of SH2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), a hematopoietic cell-specific adapter protein, is required to couple Syk family tyrosine kinase activation to downstream mediators such as phospholipase C (PLC)-gamma following TCR, platelet collagen receptor and mast cell Fc epsilon R stimulation. In addition to T cells, mast cells and platelets, SLP-76 is expressed in monocytes and macrophages. To determine the role of SLP-76 in Fc gamma R-stimulated signaling pathways in macrophages, we examined cultured bone marrow-derived macrophages (BMM) from SLP-76(-/-) and wild-type mice. In this study, we show that Fc gamma R cross-linking rapidly induces tyrosine phosphorylation of SLP-76 in wild-type BMM. Surprisingly, however, BMM from SLP-76(-/-) mice activate ERK2 and phosphorylate PLC-gamma 2 following Fc gamma R ligation. Furthermore, SLP-76(-/-) BMM display normal Fc gamma R-dependent phagocytic function and reactive oxygen intermediate production. SLP-76(-/-) and SLP-76(+/+) BMM secrete comparable levels of IL-12 in response to lipopolysaccharide and IFN-gamma. To examine macrophage function in vivo, SLP-76(-/-) mice were challenged i.v. with Listeria monocytogenes. SLP-76(-/-) mice survive and efficiently contain the acute phase of infection similar to wild-type mice but exhibit a stable chronic infection attributed to the lack of mature T cells. These data show that, although SLP-76 is required to couple Syk family PTK activity to downstream mediators and effector functions in Fc gamma R-induced pathways in some cell types, activation of Fc gamma R-dependent pathways occurs independently of SLP-76 in BM     

Increased density of HLA-DR antigen on monocytes of patients infected with the human immunodeficiency virus

Monocytes and macrophages play an important role in the pathogenesis of the acquired immunodeficiency syndrome (AIDS). Dysfunction of these cells contributes to the immunocompromised state that characterizes AIDS, and they probably serve as a reservoir for the human immunodeficiency virus (HIV). HIV-infected macrophages may also be responsible for the infection of many CD4-positive lymphocytes by means of cell to cell contact during the initiation of immunological responses. The efficiency of this process would be enhanced by activation of the macrophages, since that is accompanied by increased expression of class II major histocompatibility (HLA-DR) antigen. Using a direct blood antibody marking procedure in conjunction with flow cytometry, we have analyzed the expression of HLA-DR antigen on the surfaces of monocytes present in the peripheral blood of HIV-infected patients. In contrast to the results reported by other investigators who purified the monocytes using Ficoll-Hypaque centrifugation prior to antibody marking, we found that the percentage of monocytes expressing HLA-DR antigen was identical in the patients and normal controls. However, a subpopulation of monocytes was detected in the blood of the majority of the patients that was expressing increased levels of HLA-DR antigen. It was also found that the proportion of monocytes with a high density of HLA-DR antigen on their surfaces negatively correlated with the absolute numbers of CD4-positive lymphocytes present in the peripheral blood of the patient. These findings support the postulated role of monocytes and macrophages in the HIV infection and ultimate destruction of CD4-positive lymphocytes.     

Dengue virus neutralization is modulated by IgG antibody subclass and Fcγ receptor subtype

Severe dengue virus (DENV) infection is epidemiologically linked to pre-existing anti-DENV antibodies acquired by maternal transfer or primary infection. A possible explanation is that DENV immune complexes evade neutralization by engaging Fcγ receptors (FcγR) on monocytes, natural targets for DENV in humans. Using epitope-matched humanized monoclonal antibodies (mAbs) and stable FcγR-transfected CV-1 cells, we found that DENV neutralization by IgG1, IgG3, and IgG4 mAbs was enhanced in high-affinity FcγRIA transfectants and diminished in low-affinity FcγRIIA transfectants, whereas neutralization by IgG2 mAbs (low-affinity ligands for both FcγRs) was diminished equally. In FcγR-negative Vero cells, IgG3 mAbs exhibited the strongest neutralizing activity and IgG2, the weakest. Our results demonstrate that DENV neutralization is modulated by the Fc region in an IgG subclass manner, likely through effects on virion and FcγR binding. Thus, the IgG antibody subclass profile generated by DENV infection or vaccination may independently influence the magnitude of the neutralizing response.     

Increased expression of human monocyte HLA-DR antigens and Fc gamma receptors in response to human interferon in vivo

The expression of class II MHC encoded antigens (HLA-DR) and Fc gamma receptors by peripheral blood monocytes from untreated patients with small cell carcinoma of the bronchus was compared with that of normal donors. Fc gamma receptor expression was found to be elevated in these patients in comparison with normal. In contrast HLA-DR antigen expression by patients' monocytes was somewhat depressed in comparison with normal. Continuous intravenous infusion of a total of 400 megaunits/m2 of human lymphoblastoid interferon-alpha (IFN-alpha) over a 5 day period markedly increased both monocyte HLA-DR antigen expression and Fc gamma receptor expression in comparison with that of untreated patients and normal donors. The initial increases declined somewhat but were still evident after 3 weeks of intermittent intramuscular IFN-alpha therapy.