Monocyte-T-Cell Interactions in the Regulation of Polyclonal B-Cell Response

Human peripheral blood monocyte subsets with and without Fc receptor for human IgG are known to suppress (FcR+) and enhance (FcR-) pokeweed mitogen-induced polyclonal immunoglobulin synthesis in vitro. The ability of these subsets to modulate immunoglobulin production in the presence or absence of OKT8+ T cells and under conditions where suppressor T-cell activation was blocked by irradiation or mitomycin C was studied. It was shown that, regardless of the presence or absence of suppressor T cells, FcR+ monocytes can suppress immunoglobulin production if their number in culture exceeds 20%. However, at lower numbers this monocyte subset was suppressive only when suppressor T cells were activated. The suppressor T-cell activation was shown to be independent of the predominant presence of the FcR+ or FcR- monocyte subset. Moreover, the enhancing effect of FcR- monocytes was not caused by their interference with suppressor T-cell activation.     

T Lymphocyte Recognition of Alloantigen in Vitro

The present experiments were carried out in order to answer the question whether the precursor T cells of cytotoxic T lymphocytes (PCTL) are Fc receptor positive (FcR+), Fc receptor negative (FcR-), or both. The data show that cytotoxic T lymphocytes (CTL) can be generated in vitro from both FcR+ and FcR- PCTL. Furthermore, we investigated if the function of FcR+ and FcR- CTL differed in the allograft response in vitro. Qualitatively, FcR- PCTL differentiate into FcR- CTL only, whereas FcR+ PCTL differentiate into both FcR+ and FcR- CTL. However, quantitatively, CTL generated from FcR- PCTL display a higher T cell mediated cytotoxicity than CTL generated from FcR+ PCTL. Mixing experiments indicate that FcR+ T cells regulate the differentiation of FcR- PCTL into CTL. These conclusions hold true for PCTL in both normal and memory responder cell populations.     

Immunoregulation of lymphoproliferation in vitro by monocytes and their subpopulations. I. The induction of suppressor T cells in long term cultures and the role of MHC class II determinants and preliminary characteristics

Peripheral blood monocyte (MO) subpopulations isolated on a basis of functional expression or a lack of Fc receptor (FcR+ and FcR- MO) were used to study the regulation of the antigen (PPD)-driven lymphoproliferation. Long-term cultures of T lymphocytes with FcR+ MO, but not FcR- MO, led to the induction of T suppressor (Ts) cells that inhibited antigen-driven lymphoproliferation in the test cultures. These Ts cells were resistant to mitomycin C, belonged to afferent-acting category of Ts cells, expressed CD8 and HLA-DR determinants, and showed no antigenic specificity nor genetic restriction in action. The expression of MHC class II molecules (HLA-DR and HLA-DP but not HLA-DQ determinants) on MO which were used for antigen presentation was critical for Ts cell induction. It was concluded that specialized MO subpopulations may regulate the lymphoproliferation by inducing Ts cells (or Ts cell circuit) that in turn inhibit antigen-driven immune response.     

Immunoregulation of lymphoproliferation in vitro by monocytes and their subpopulations. II. Phenotypic changes of T cells in cultures activated with antigen and mitogen.

The aim of this study was to analyze phenotypes of T cells activated by mitogen (PWM) and antigen (PPD) in the presence of FcR+ or FcR- monocytes. It was found that CD4+ and CD8+ lymphocytes are preferentially activated in the presence of different monocyte subpopulations. Expression of HLA-DR and CD25 on CD4+ lymphocytes was greater in cultures activated in the presence of FcR-. CD8+ lymphocytes were more efficiently activated (expression of HLA-DR) when FcR+ monocytes were added to culture. In the presence of FcR+ monocytes an increased expression of CD45RA antigen on CD4+ cells was also observed. These data support our previous functional studies which showed that "suppressor" T cells of CD8+ phenotype are activated in the presence of FcR+ monocytes.     

FcR+ and FcR- monocytes differentially secrete monokines during pokeweed mitogen-induced T-cell-monocyte interactions.

Monocyte subpopulations which differ in the expression of Fc receptor for human IgG (FcRI) differentially regulate the T-cell-dependent, pokeweed mitogen (PWM)-induced, polyclonal B-cell response. We, thus, studied the cytokine production in human peripheral blood monocyte and T-lymphocyte cultures activated with this lectin. Monocytes or their FcR+ and FcR- subpopulations stimulated with PWM were cultured with or without T lymphocytes or their CD4+ and CD8+ subsets. Both monocyte subpopulations cultured alone produced similar amounts of tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), but FcR- monocytes showed significantly enhanced ability to secrete interleukin-1 (IL-1). T cells, especially CD4+, added to monocyte cultures enhanced IL-1 production. This enhancement was presumably due to interferon-gamma (IFN-gamma) release by T lymphocytes, since this lymphokine enhanced IL-1 secretion when added to PWM-stimulated cultures of monocytes. Addition of monocytes, in particular the FcR+ subpopulation, greatly enhanced production of IFN-gamma by T lymphocytes. Although both T-cell subsets produced IFN-gamma, the CD4+ cells were more efficient. These results indicate that in PWM-stimulated cultures subpopulations of monocytes differ in secretion of cytokines, which might explain their differential effect on T-cell-dependent immune responses in vitro.     

Monocyte subsets in the production of inhibitory factor by Candida albicans-activated human T cells.

Macrophages are essential for the proliferative response of human T lymphocytes to a purified polysaccharide extract from Candida albicans (MPPS). The role of macrophages as antigen-presenting cells in the production of an antigen non-specific inhibitory factor (nsINH) by MPPS-activated T cells was also investigated. Fc receptor positive (FcR+) or negative (FcR-) plastic-adherent mononuclear cells were used as MPPS-presenting cells, and the results show that the FcR- subset is mainly responsible for the release of nsINH from activated T cells.     

Immunological properties of Fc receptor on lymphocytes. VIII. The behaviour of FcR+ and FcR- T cells in cell-mediated immune responses.

The roles of splenic FcR+ and FcR- T cells from mice immunized either with allogeneic cells or with LCMV or stimulated either with MMC-treated allogeneic cells or TNP-modified syngeneic cells in vitro were examined for cell-mediated cytolytic responses. Effective lysis was observed in the FcR- cell fraction enriched with nylon wool eluted T cells in all experiments. Killer cells were generated from the FcR- T-cell fraction after exposure to either allo-, virus- or chemically modified antigen. On the other hand, the lytic activity of the FcR+ T-cell fraction, was low but nevertheless still significant. However, this weak cytotoxicity was increased by 24 h of incubation, although the recovery of living cells was found to be significantly lower in the FcR+ than in the FcR- T-cell fraction. This suggested that non-killer cells, which could interfere with the activity of pre-killer cells, were perhaps preferentially eliminated after binding with the immune complexes. Moreover, almost complete inhibition of lytic activity was achieved in allo-activated FcR+ T, but not FcR- T-cell fraction, after they had been treated with immune complexes, implying a functional significance of FcR in the manifestation of lytic activity.     

The regulation of polyclonal immunoglobulin synthesis by FcR+ and FcR- monocyte subsets

FcR+ and FcR- monocyte subsets were added to the pokeweed mitogen (PWM) or Staphylococcus aureus Cowan I-stimulated cultures of peripheral blood mononuclear cells (PBMC) or to PBMC depleted of monocytes. The numbers of immunoglobulin-secreting cells (ISC) and cells with intracytoplasmic immunoglobulins (PC) were evaluated 6 days later. The addition of FcR- subset increased the number of ISC in cultures of PBMC stimulated with PWM and reconstituted the response of monocyte depleted PBMC. In contrast, FcR+ monocytes suppressed PWM-induced response and, when added in high dose, also that induced by S. aureus. The FcR+ monocytes suppressed the response by inhibition of immunoglobulin secretion but not the development of PC. This suggests that FcR+ monocytes may modulate humoral response by preferential inhibition of the final differentiation of B lymphocytes into ISC.     

Syngeneic mixed lymphocyte reactions to murine Fc receptor-bearing, nonadherent low density lymph node cells and epidermal Langerhans cells

Both Langerhans cells from BALB/c mouse epidermis and nonadherent, low density (LD) cells, obtained from collagenase-treated minced lymph node, are stimulatory for isolated T cells in syngeneic mixed lymphocyte reaction (SMLR). The method of preparation of LD cells influences whether Fc receptor (FcR) can be detected on them. Fc receptors on nonadherent LD lymph node cells can be detected only if the same procedure is employed as that for Langerhans cell isolation, i.e., Ig-free bovine albumin must be used during gradient centrifugation and EA rosetting must be done overnight at 4 degrees C. Thus, both FcR+ and FcR- LD lymph node cells, as well as FcR+ Langerhans cells, stimulate SMLR. Although Ig+ cells in the LD fraction also stimulate the SMLR, their removal does not affect the stimulating capacity of the LD lymph node fraction.     

The Fc Receptors of Normal and Cancer Patients Monocytes

The ability of human monocytes from normal donors and gastric-cancer patients to form rosettes with ?0? Rh⁺(D) human erythrocytes coated with hyperimmune IgG anti-D antibody (EA_hu) and to kill the same target in antibody-dependent cellular cytotoxicity (ADCC) were assessed. Trypsin pretreatment of normal monocytes decreased their ability to form rosettes with EA_hu complexes, but their ADCC activity was unaffected. The Fc receptor (FcR) expression and ADCC activity of monocytes of cancer patients were elevated, and trypsin-treatment led to their further increase. The elevated values were related to the presence of the tumour. These results may suggest that human monocytes possess trypsin-sensitive and trypsin-resistant Fc receptors. The trypsin-resistant FcR seems to be involved in ADCC phenomenon and to be preferentially expressed on monocytes of some cancer patients.     

IgG3-mediated enhancement of the antibody response is normal in Fc gammaRI-deficient mice

Antibodies, administered together with their specific antigen, can feedback-regulate antibody responses to this antigen. IgG1, IgG2a and IgG2b enhance antibody responses to soluble protein antigens. This effect is primarily mediated by FcRs as enhancement is impaired in FcR gamma-/- mice, reported to lack Fc gammaRI and Fc gammaRIII because of deletion of the common FcR gamma chain. Also IgG3 can enhance antibody responses. However, this effect is unperturbed in FcR gamma-/- mice but severely impaired in complement-depleted animals and in animals lacking complement receptor 1 and 2. Although this argues against involvement of Fc gammaRs, FcR gamma-/- mice may express one-fifth of the normal levels of Fc gammaRI and, in addition, Fc gammaRI has been suggested to bind IgG3. We re-investigated the dependence of IgG3-mediated enhancement on Fc gammaRs using a mouse strain selectively lacking Fc gammaRI and found that IgG3-mediated enhancement is completely normal. Unlike IgE and IgG2a, which are both thought to enhance T-cell proliferation via FcR-mediated antigen presentation, IgG3 was a poor enhancer of T-cell proliferation both in vivo and in vitro. These findings argue against a significant involvement of Fc gammaRs in IgG3-mediated enhancement of antibody responses and support our previous conclusion that complement plays a major role.     

Mycobacterial-induced cytotoxic T cells as well as nonspecific killer cells derived from healthy individuals and leprosy patients

Little information is available about the generation and specificity of the cytotoxic cells that eliminate human monocytes/macrophages infected with mycobacteria. To address this we have developed a cytotoxicity assay in which 51Cr-labeled monocytes pulsed with bacillus Calmette Guerin (BCG) or Mycobacterium leprae, were used as target cells in overnight cytotoxicity assays. As effector cells, peripheral blood mononuclear cells from healthy occupational contacts or from leprosy patients stimulated with antigen for 7 days were used. Cytotoxicity against antigen-pulsed monocytes that could be induced by mycobacterial antigens was proportional to the degree of antigen responsiveness in each individual, as measured in lymphocyte transformation tests. The lepromatous leprosy patients tested were often poor responders to BCG as well as M. leprae, both with regard to induction of cytotoxicity as well as in lympho-proliferation. Killing was significantly higher against antigen-pulsed vs. nonpulsed monocytes, although significant killing was induced against the latter as well and paralleled by induction of natural killer activity against the K-562 target cell. Cross-reactivity was observed between BCG and M. leprae, but not with unrelated antigen (tetanus toxoid) or with endogenous stress proteins induced by heat shock. M. leprae- and BCG-activated cytotoxic cells were found in both the CD4-CD8+ and CD4+CD8- populations, whereas in contrast the soluble antigen, purified protein derivative of M. tuberculosis, generated cytotoxic cells that were exclusively of the CD4+ phenotype. The involvement of both specific T cells as well as nonspecific cells in the killing of human macrophages may be important with respect to protection and immunopathology induced by mycobacterial antigens.     

Fcγ receptor II dependency of enhanced presentation of major histocompatibility complex class II peptides by a B cell lymphoma

Here we show that the B cell lymphoma A20.292 is capable of enhanced antigen presentation to CD4⁺ T cells in the presence of specific antibodies. This enhancement was inhibited by anti-Fcγ receptor (R) antibodies, suggesting that it might be due to preferential uptake of the antigen/antibody complex through the FcγRII receptor. However, immunoprecipitation studies revealed that the FcR of A20.292 cells was of the B cell type, FcγRIIb1, which is not thought to be able to internalize antigen/antibody complexes via clathrin-coated pits. It was considered unlikely that A20.292 had an altered form of the B cell FcγR (RIIb1) receptor that enabled internalization, since similar enhancing effects were also observed using an FcγRII^? cell line that had been transfected with FcγRIIb1. To reconcile these findings with the expression of FcγRIIb1, it is postulated that immune complexes are concentrated on the cell surface by the FcγRIIb1 and are thus available for preferential uptake by random fluid-phase endocytosis. This results in more efficient generation of the epitopes recognized by these T cell hybridomas.     

Natural suppressor cells in human leprosy: the role of HLA-D-identical peripheral lymphocytes and macrophages in the in vitro modulation of lymphoproliferative responses

Six families with HLA-D-identical siblings suffering from leprosy were studied. Lymphocytes and macrophages isolated from the peripheral blood were co-cultured with allogeneic, HLA-D-identical cells and stimulated with M. leprae antigens and concanavalin A. Tuberculoid patients had circulating lymphocytes which showed marked functional suppression of lymphoproliferative responses to antigen and mitogen. In contrast, lepromatous patients showed weak lymphocyte suppressor activity. Macrophages derived from responder individuals augmented, while those derived from lepromatous patients inhibited, M. leprae-induced proliferation of lymphocytes.     

Immunological heterogeneity of human monocyte subsets prepared by counterflow centrifugation elutriation.

Human peripheral blood mononuclear cells were separated into three fractions by means of counterflow centrifugation elutriation (CCE). The first fraction, eluted at a flow rate of 24 ml/min, was composed of lymphocytes with less than 2% contaminating esterase-positive cells. The cells in this fraction were incapable of responding to either soluble antigen (tetanus toxoid) or particulate antigen (cytomegalovirus-infected fibroblasts) unless recombined with accessory cells. The second fraction, eluted at a flow rate of 28 ml/min, was composed predominantly (72%) of small Ia, leu M3, and esterase-positive monocytes, which stained weakly with leu 10 antibody. Cells in this fraction efficiently presented soluble and particulate antigens to monocyte-depleted lymphocytes. Of the remaining cells, 87% were large esterase-positive monocytes that labelled strongly with Ia, leu M3, and leu 10. These cells were less efficient in antigen presentation than the small monocytes. However, lymphocytes activated with antigen-pulsed large monocytes exhibited more suppressor cell activity than those activated with antigen-pulsed small monocytes.     

Analysis of Fcγ receptors on human peripheral blood leukocytes by flow microfluorometry. I. Receptor distributions on monocytes,Tγ cells and cells labeled with the 3A1 anti-T cell monoclonal antibody

A dual parameter flow microfluorometric technique for accurately measuring Fcγ receptor (FcR) expression on defined subsets of cells within a heterogeneous cell sample was developed. The FcR distribution of human peripheral blood mononuclear cells consists of three distinct peaks. By analyzing cells fluorescently labeled with the 3A1, an anti-T cell hybridoma antibody (using a green-emitting fluorophore) and for FcR (with a red-emitting fluorophore), and by using cell isolation procedures, it was shown that the cells lying within the peak with intermediate FcR density are mainly monocytes, while cells lying within the peaks with highest and lowest (i.e. negative) FcR densities are predominantly T cells. The FcR⁺ T cells (T_γ cells) express higher levels of the 3A1 antigen than other T cells, thus demonstrating the utility of the 3A1 hybridoma antibody as a marker for T_γ cells.     

In vitro suppression of interleukin 2 production by Mycobacterium leprae antigen.

The suppressive activity of three different lots and sources of Mycobacterium leprae (M. leprae) was studied by measuring the inhibitory effect on interleukin 2 (IL-2) production in normal subjects. All three M. leprae preparations had suppressive activity on IL-2 production when peripheral blood mononuclear leucocytes (PBML) were stimulated with the mitogens PHA-P or Con A in a dose response. M. leprae also had suppressive activity on IL-2 production when PBML were stimulated with the specific antigen, PPD. The inhibitory activity of M. leprae on IL-2 was not due to the direct interaction of M. leprae and IL-2 because direct mixing of IL-2 with different concentrations of M. leprae did not alter the activity of IL-2. Incorporation of M. leprae for 0, 6 and 12 h in PHA-P and PBML cultures had no inhibitory effect on IL-2 production; however, after 14, 16 and 18 h of M. leprae incorporation, significant inhibitory effects were noted on IL-2 production. The suppressive mechanism of M. leprae was studied by incorporating M. leprae into PBML or adherent cells. The suppressive activity could be detected in both M. leprae-stimulated PBML and M. leprae-stimulated monocyte supernatant fluids. The suppressive mechanism of M. leprae was further evaluated by incorporating 1 and 2 micrograms/ml of indomethacin in PBML containing PHA-P and M. leprae. The suppressive activity of M. leprae was significantly diminished by indomethacin, suggesting that the inhibitory effect of M. leprae may result from the induction of PBML and adherent cells to produce the immunosuppressive activity of prostaglandin(s).     

Defective Immune Complex Degradation by Monocytes in Patients with Systemic Lupus Erythematosus.

The binding of 125I-labelled anti-bovine serum albumin (BSA)-BSA immune complexes (IC), giving a final molar antibody to antigen ratio of 1:1, to monocytes isolated from 18 patients with systemic lupus erythematosus (SLE) and from 10 normal healthy donors was quantitatively investigated. The degradation of the bound IC by the same monocytes was kinetically determined at the same time. The assays were performed on monocyte monolayers. Scatchard plots at 4 degrees C demonstrated that monocytes from patients with active SLE expressed a mean Fc gamma receptor (FcR) number that was 22% higher than that of the controls, although this did not reach statistical significance. The FcR number of normal monocytes and the degradation rate of anti-BSA-BSA complexes by the same cells showed a positive correlation. At the same time, the digestion of anti-BSA-BSA complexes by monocytes of SLE patients with active disease was prolonged, despite their enhanced FcR-ligand binding. The dissociation of FcR-ligand binding and FcR-mediated degradation suggests that the IC degradation is controlled by altered biochemical mechanisms in the monocytes of SLE patients.     

Characterization of Two Fc Receptors for Mouse Immunoglobulins on Human Monocytes and Cell Lines

We have previously reported a polymorphism in the mitogenic effect of murine (m) IgG1 anti-CD3 monoclonal antibodies. This polymorphism was genetically determined and could be attributed to polymorphism of the Fc receptor (FcR) for mIgG1 present on human monocytes. We have now extended these studies by quantitating FcR expression on monocytes and cell lines by a recently developed EA rosette assay, using the erythrocyte-associated pseudoperoxidase activity. The data show that the polymorphism of the monocyte FcR for mIgG1 is based on a quantitative rather than an absolute difference. Furthermore, this FcR is specific for mIgG1 and does not bind mIgG2a or mIgG2b nor, surprisingly, human IgG. The expression of this FcR on cell lines correlates with their accessory function in IgG1 anti-CD3-induced T cell proliferation. mIgG2a can inhibit the rosetting of monocytes with erythrocytes sensitized with human IgG. The FcR detected by this rosette technique can interact with all four human IgG subclasses but not with mIgG1 or mIgG2b. The expression of this type of FcR on human cell lines correlates well with their ability to support mIgG2a anti-CD3-induced mitogenesis. These direct measurements of FcR expression support the concept that human monocytes have two independent FcR with affinity for mouse IgG: one receptor specific for mIgG2a (which also binds human IgG), and a second specific for mIgG1.