B lymphocyte regulation of the immune system. II. Inhibition of Fc receptor expression of lymphocytes by BEF, a lymphokine of B cell origin

Recently, we described a new lymphokine of B cell origin, capable of selectively preventing the differentiation of T suppressor cells from the precursor into the effector stage. As a result, antibody production against various antigens is markedly increased. We termed this lymphokine B cell-derived enhancing factor (BEF). To discern the mechanism(s) by which BEF interferes with the activation of T suppressor cells, experiments were undertaken to explore the effect of BEF on the induction of Fc receptors (FcR). The induction of FcR on T cells has been implicated in the down-regulation of antibody synthesis, and it has been suggested that the expression of FcR for a given immunoglobulin precedes the release of factors with regulatory functions for the corresponding isotype. In the experiments reported here, murine spleen cells were incubated for 24 hr in the presence of IgG1 or IgA monoclonal antibodies, were washed, and the number of FcR gamma 1+ and FcR alpha+ cells were calculated by a rosette assay. The effect of BEF was studied either during the inductive phase or before, i.e., by pretreating the cells with BEF for 18 hr at 37 degrees C before the inductive phase. Our results show that BEF abolishes, in a dose-dependent manner, the expression of isotype-specific FcR in spleen cells when present during the inductive phase, as well as when cells are pretreated with it. In successive experiments, we tested the effect of BEF on the induction of FcR on T cell-enriched or B cell-enriched spleen cells. The results show that BEF is effective in selectively inhibiting FcR expression on T lymphocytes, but not on B lymphocytes, once isolated from the total spleen cell population. These findings provide further insight into the mechanism by which BEF modulates the immune response, and suggest that different mechanisms may be involved in the induction of FcR on T and B lymphocytes, respectively.     

Activation of T lymphocytes by the Fc portion of immunoglobulin

T lymphocytes are stimulated to release T-cell-replacing factors in response to Fc fragments of human IgG. Lyt 1⁺23^? T cells are directly triggered to factor production by Fc subfragments, derived from intact Fc fragments by macrophage-dependent enzymatic cleavage. These factor(s) replace T cell function in two Fc-mediated immune responses; induction of polyclonal antibody synthesis, and potentiation of anti-SRBC responses.     

Cellular cooperation in mitogen-induced human leukocyte inhibitory factor (LIF) synthesis.

Interactions between human T and B lymphocytes and between lymphocyte subpopulations and accessory cells in lymphokine synthesis were investigated. The cells were stimulated with leukoagglutinin (LA), concanavalin A (Con A), protein A (prot A) and anti-β₂-microglobulin (anti-β₂m). The presence of leukocyte inhibitory factor (LIF) in the culture supernatants was tested by the agarose-migration method. The results indicated that monocytes augmented LIF synthesis of T cells but suppressed that of B cells. Monocyte-helper effect was mediated by both cell-cell contact and soluble factors. In addition, T lymphocytes were found to augment B-cell LIF production. B lymphocytes enhanced Con A- but suppressed LA-induced LIF production by T cells. T-cell/B-cell collaboration was based on a direct cell-cell contact and no soluble factors were found.     

The mechanisms of antibody-dependent killing mediated by lymphoid and myeloid cells are distinct based on different divalent cation requirements

To further understand the mechanism(s) of antibody-dependent cell-mediated cytotoxicity (ADCC) by various effector populations, we have examined the extracellular Ca++ and Mg++ requirements for ADCC performed by lymphocytes, monocytes, polymorphonuclear leukocytes and peritoneal macrophages. We have used the anti-Fc gamma R-bearing hybridoma cell lines (HC) as self directed targets for ADCC to analyse the triggering ability of each of the three defined Fc gamma R; Fc gamma RI, Fc gamma RII, and Fc gamma RIII. Lymphocyte killing of the anti-Fc gamma RIII bearing HC (HC 3G8) was Ca++ dependent, but Mg++ independent. In contrast, monocytes and PMN killed the anti-Fc gamma RI- (HC 32) and the anti-Fc gamma RII- (HC IV.3) bearing HC in a Mg++-dependent, Ca++-independent fashion. In addition, freshly prepared monocytes were able to kill HC 3G8 in a Mg++-dependent, Ca++-independent fashion, indicating that low levels of Fc gamma RIII may be functionally detected on monocytes. Peritoneal macrophages were able to kill all three of the anti-Fc gamma R bearing HC in a Mg++-dependent, Ca++-independent fashion. Thus, the same target is lysed by myeloid cells in the presence of Mg++ without Ca++ and by lymphoid cells in the presence of Ca++ without Mg++. These results suggest that at least two distinct mechanisms of ADCC exist that depend on the type of effector cell mediating antibody-dependent killing and not necessarily on the Fc gamma R type triggered.     

Interferon production by human and murine lymphocytes in response to alloantigens

The production of interferon (IF) by human and mouse lymphocytes sensitized to alloantigens in mixed lymphocyte cultures (MLC) was analyzed. During primary MLC, IF appeared in the culture fluid on day 2 and was maximal on day 5. Based on several biologic criteria, the IF produced is of the "immune" type. When lymphocytes sensitized to alloantigens were reestimulated in vitro, IF was produced within a few hours of culture. In all stimulated cultures, cell proliferation was observed in spite of the high concentrations of IF. The IF-producing cells in human MLC were identified as T lymphocytes lacking the receptor for the Fc fragment of IgG molecules (Fc gamma R(-)). Human MLC supernatants containing immune type IF mediate the enhancement of natural killer (NK) cell activity and protect NK target cells from lysis.     

Complement-dependent stimulation of normal lymphocytes by immune complexes.

Normal rabbit lymphocytes were stimulated to proliferate in vitro by antibody-antigen complexes. Stimulation was dependent upon C activity. Heat-inactivation or zymosan-treatment of the serum used in culture caused a 75 to 100% loss of responsiveness to the complexes. Serum-free culture or cultures with less than 1% serum supported only low levels of stimulation, but responsiveness reappeared proportionally with increased serum concentration. The low level dose-dependent responses seen in the absence of active C may have been due to C carried over with the cells or to stimulation independent of C. Aggregated rabbit gamma-globulin tested over a broad dose range failed to stimulate normal lymphocytes more than minimally whether or not C was present. Stimulation with immune complexes was sustained by C4-deficient guinea pig serum, indicating participation of the alternative C pathway. Normal rabbit lymphocytes from peripheral blood, bone marrow, spleen, and lymph node proliferated in response to rabbit antibody-antigen complexes. Normal thymocytes were consistently unresponsive to the complexes.     

Fc gamma-receptor-bearing, non-B lymphocytes in human peripheral blood: cytophilic immunoglobulin binds almost exclusively to large granular lymphocytes

Cytophilic IgG (CYT-Ig) has previously been reported to bind to both the "TG" (E+, Fc gamma R+) and "L" (E-, Fc gamma R+) subsets of non-B lymphocytes in human peripheral blood. Present investigations show that IgG-binding cells, as detected by a sensitive antiglobulin rosetting reaction, are contained almost entirely within the large granular lymphocyte (LGL) subpopulation, and that fewer than 5% of other non-B lymphocytes acquire IgG from serum. Cell membrane-bound IgG sterically blocks the reaction of LGL with sheep red blood cells and therefore influences the proportions of these cells characterized as TG (E+) or L (E-) lymphocytes. Although the majority of TG lymphocytes are LGL, a further subpopulation of E+, Fc gamma R+ cells are detectable under particular test conditions. Unlike LGL, these lymphocytes do not react with rabbit IgG-coated ox RBC (EAG) in saline, but will form EAG rosettes when the reaction is enhanced in the presence of Ficoll. These Fc gamma R+ cells are mostly of typical small-lymphocyte morphology and do not bind detectable amounts of CYT-Ig, nor do they express the monoclonal antibody-defined VEP 13 determinant associated with Fc gamma R on LGL.     

Proliferation of murine thymic lymphocytes in vitro is mediated by the concanavalin A-induced release of a lymphokine (costimulator).

Mitogen-induced proliferation of lymphocytes may in theory result directly from the interaction of mitogen with the cells, or indirectly as a result of the mitogen-stimulated release of lymphokines. In the case of murine thymic lymphocytes exposed to concanavalin A (Con A) in tissue culture, we have determined that mitogenesis depends upon a lymphokine. Interaction of the thymic lymphocytes with lectin is necessary, but not sufficient, for mitogenesis. A lymphokine, or costimulator for mitogenesis, is released by normal spleen or thymus cells during the first 16 hr of their exposure to Con A, and in the presence of a phytomitogen it stimulates thymic mitogenesis. Under conditions of low costimulator levels, no mitogenesis follows the interaction of Con A with cells. The response of adult CBA/J mouse thymocytes to phytohemagglutinin (PHA) is very low, compared to their response to Con A. When costimulator is added to PHA, the cells respond as well as they do to Con A. Costimulator does not act through Con A-binding sites on thymus cells. Its production is dependent on both cells carrying omega surface antigen (T lymphocytes) and adherent cells of the macrophage-monocyte series. The adherent population, but not the T cells, may be heavily irradiated without affecting production of costimulator. Costimulator is not a mitogen on its own.     

Fc gamma 2b receptor-mediated prostaglandin synthesis by a murine macrophage cell line (P388D1)

The nature of signals transmitted by two types of Fc gamma receptors (one specific for IgG2b and the other for IgG2a) present on the surface of a murine macrophage cell line (P388D1) was investigated. Specific binding of IgG2b (presented as EA2b) to cell surface Fc gamma 2br triggered the release of 3H-arachidonic acid and 3H-prostaglandins (PG) from P388D1 cells that were prelabeled with 3H-arachidonate. The release of 3H-arachidonic acid, which increased in a dose-dependent manner, was enhanced by exogenous Ca++ (1.25 mM) and was completely blocked by ethylenediaminetetraacetate (EDTA) (4 mM) or a phospholipase A2 inhibitor, p-bromophenacylbromide (7 microgram/ml). A cyclooxygenase inhibitor, indomethacin (9 microgram/ml), reduced the 3H-arachidonic acid release and completely blocked the conversion of arachidonate into PG. Cytochalasin D (1 microgram/ml), which inhibited the phagocytosis of immune complexes by 90% of P388D1 cells, did not affect the Fc gamma 2bR-triggered release of arachidonic acid. Specific binding of IgG2a (presented as EA2a) to cell surface Fc gamma 2aR did not trigger the release of either 3H-arachidonic acid or 3H-PG from P388D1 cells. Our data demonstrate a signal for the activation of the arachidonic acid metabolic cascade is transmitted by Fc gamma 2bR, but not by Fc gamma 2aR, on the surface of P388D1 cells, probably through the initial activation of the phospholipase A2 activity associated with Fc gamma 2bR.     

Human monocytes, B lymphocytes, and non-B lymphocytes each have structurally unique Fc gamma receptors

Human Fc gamma-binding macromolecules were isolated from subpopulations of mononuclear cells by repetitive affinity chromatography. Mononuclear cells, nylon wool-filtered cells, plastic-nonadherent cells, and plastic-adherent cells from normal donors were radiolabeled by using 125I and lactoperoxidase. Washed cells were solubilized in 1% NP-40 buffer containing proteinase inhibitors at 0 degrees C. Fc gamma receptors were purified on human IgG-Sepharose columns by use of the repetitive affinity chromatography procedure. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated only a 52,000 to 58,000 Mr Fc gamma receptor from nonadherent cell populations. Both rosetting and nonrosetting subpopulations of non-B lymphocytes expressed the 52,000 to 58,000 Mr receptor. The predominant Fc gamma receptor isolated from plastic-adherent cells was a 60,000 to 68,000 Mr macromolecule. Cell preparations enriched in B lymphocytes yielded prominent 43,000 Mr Fc gamma receptors. Thus human monocytes, B lymphocytes, and non-B lymphocytes each appear to have structurally distinct and unique Fc gamma receptors.     

Surface immunoglobulins as targets for anti-immunoglobulin-dependent cell-mediated lysis of B cells

Human K cells are able to lyse human lymphoblastoid B cells in the presence of specific anti-human immunoglobulin isotype antibodies. The human lymphoblastoid B-cell line DAUDI (surface mu- and kappa-chains positive) was lysed in the presence of anti-mu and anti-kappa antisera, or IgG fraction of these antisera, and B-cell-depleted human lymphocytes. Lysis was not induced by anti-isotypic antisera alone or human lymphocytes alone. Lysis was not induced by antisera directed at isotypes which were not expressed on the DAUDI cells. The human lymphoblastoid B-cell line RAJI, which does not express surface membrane isotypes, could not be lysed by the anti-isotype-dependent cell-mediated mechanism. Lysis of DAUDI cells by this mechanism was mediated by Fc gamma receptor-bearing human non-B lymphocytes and required an intact Fc piece in the inducing anti-isotypic antibody. These observations are discussed as a possible role for K cells in the regulation of immune responses by interaction between anti-idiotypic antibodies and idiotype-bearing cells.     

Functions of the various IgG Fc receptors in mediating killing of Toxoplasma gondii.

The three types of IgG FcR (Fc gamma RI, Fc gamma RII, Fc gamma RIII) on human leukocytes play an important role in elimination of antibody-coated infectious agents. To further understand the role of the different Fc gamma R in mediating this killing, we examined the ability of human myeloid and lymphoid cells to kill the protozoan Toxoplasma gondii in the presence of antitoxoplasma IgG or bispecific antibodies. Although human myeloid cells (monocytes, macrophages, neutrophils, and eosinophils) all lysed unsensitized T. gondii, killing by these cells was significantly enhanced by opsonization with antitoxoplasma rabbit IgG. Human lymphocytes, however, did not lyse T. gondii unless the parasites were coated with antibody. The role of antibody and Fc gamma R in mediating ADCC of T. gondii was then examined using bispecific antibodies made by chemically cross-linking Fab fragments of antitoxoplasma antibodies to Fab fragments of antibodies specific for human leukocyte surface Ag, including Fc gamma R. Thus, simultaneous binding of these bispecifics to parasites and effector cells allowed an evaluation of killing when T. gondii were targeted to each Ag independently. Bispecifics which targeted T. gondii to Fc gamma RI, II or III enhanced lysis by monocytes. However, similar results were obtained with bispecifics targeting T. gondii to non-Fc gamma R Ag (CD11b or beta 2-microglobulin) on monocytes. Likewise, polymorphonuclear leukocytes mediated significantly more lysis in the presence of bispecifics linking T. gondii to Fc gamma RII, Fc gamma RIII, or the two non-Fc gamma R Ag CD11b and beta 2-microglobulin. Thus, although human myeloid cells did not require antibody-Fc gamma R triggering to kill T. gondii, antibody appeared to enhance lysis by capturing and directing the parasites to the effector cell surface. Human lymphocytes, in contrast, mediated significant lysis of T. gondii only in the presence of bispecifics targeting T. gondii to Fc gamma RIII, indicating a requirement for specific triggering of Fc gamma RIII for killing by large granular lymphocytes. Consequently, using bispecifics to compare targeting to specific Ag, both non-Fc gamma R and Fc gamma R, allowed determination of the role of antibody-Fc gamma R interactions in T. gondii killing. In addition, these studies demonstrate the potential of bispecifics in determining the role of specific Ag in killing of or infection by pathogens.     

Elaboration of lymphotoxin by freshly isolated human T lymphocytes and continuous lymphoid-cell lines.

Freshly isolated human T lymphocytes were demonstrated to produce lymphotoxin (LT) after mitogenic stimulation with phytohemagglutinin (PHA). In contrast, freshly isolated B lymphocytes, stimulated with two B-cell mitogens [pokeweed mitogen (PWM) and Staphylococcus protein A (Staph A)] did not produce the lymphokine, although thymidine incorporation was increased in these cells. We also examined a series of nine continuous human lymphoid-cell lines with B-cell markers and observed the spontaneous release of either large or small amounts of cytotoxin, or none at all. Cytotoxin from one of the productive cell-lines (H4218) was compared in detail with that obtained from PHA-stimulated, freshly isolated human lymphocytes. The behavior of the two cytotoxins was found to be identical in respect to migration on polyacrylamide gel, neutralization with rabbit anti-human α-LT serum, ultracentrifugation on 5–30% sucrose gradients, and stability for 15 min at 75 °C. Observation of these identical parameters strongly suggests that the α-LT elaborated by PHA-stimulated, freshly isolated human lymphoid cells is the same as the cytotoxin obtained from the continuous human lymphoid-cell line H4218. Thus α-LT may also be produced in quantity from continuous lymphoid-cell lines by mass tissue-culture techniques, which are more readily applicable to large-scale production than is purification from freshly cultured human lymphoid tissues. Notably, in cultures of freshly isolated human lymphoid cells, only T cells, and not B cells, generated lymphotoxin. However, continuous human lymphoid-cell lines with B-cell markers can also secrete this lymphokine.     

Augmentation of macrophage complement receptor function in vitro. V. Studies on the mechanisms of ligation of macrophage Fc receptors required to trigger macrophages to signal T lymphocytes to elaborate the lymphokine that activates macrophage C3 receptors for phagocytosis

We previously described a unique lymphokine that activates macrophage C3 receptors for phagocytosis. The lymphokine is generated when T lymphocytes receive a signal from macrophages that have ingested IgG-coated material. In the present work, we examined the mechanisms by which macrophage Fc receptors must be engaged for macrophages to signal lymphocytes to elaborate the lymphokine. We found that ingestion mediated by any of the three classes of murine macrophage Fc receptors was sufficient to trigger macrophages, and that engagement of macrophage Fc receptors by immobilized immune complexes was effective as well. We also found that ligation of Fc receptors by an anti-Fc receptor IgG antibody or by its F(ab')2 or Fab fragments also triggered macrophages. The ability of monovalent ligation of the receptor to elicit biologic activity suggests that this system may be of value in elucidating general mechanisms by which ligand binding of receptors is transduced into biologic effects.     

Enhancement of antigen-induced interleukin 4 and IgE production by specific IgG1 in murine lymphocytes.

Conalbumin (CA)-specific type 2 helper T cell (Th2) clone, D10G4.1 (D10) produces IL4 when stimulated with varying doses of TNP-CA in the presence of mitomycin C-treated C3H spleen cells or purified B cells as antigen-presenting cells (APC). The production of IL4 was assessed by bioassay and by expression of IL4 mRNA. IL4 production reached maximum at 100 micrograms/ml of TNP-CA, whereas 1 microgram/ml of the antigen induced less than 10% of the maximum level of IL4. This lower level of IL4 production was augmented to the maximum level when monoclonal anti-TNP IgG1 was added to the culture at 0.5-1 microgram/ml. Anti-TNP IgE, but not anti-TNP IgM, was also effective, though IgE was 1/10 as effective as IgG1. IgG1 with an irrelevant specificity and F(ab')2 of anti-TNP IgG1 did not show augmenting effects. Moreover, the enhancement by anti-TNP IgG1 was completely abolished by monoclonal antibody against murine Fc gamma RII, 2.4G2. These results suggest that a low dose of the antigen complexed with IgG1 is focused on APC by means of Fc gamma RII, processed, and presented efficiently to the Th2 clone. On the other hand, the co-culture of D10 with normal C3H B cells in the presence of 1-100 micrograms/ml TNP-CA resulted in polyclonal IgE production. Anti-TNP IgG1 markedly augmented the lower level of IgE production induced by a suboptimal dose of the antigen (1 microgram/ml). This augmentation was shown to be dependent on endogenous IL4 because the enhancement was abolished by monoclonal anti-IL4 (11B11).     

Further evidence for heterogeneity of Fc gamma-receptors on guinea pig splenic B and T lymphocytes: analysis using monoclonal antibodies to two distinct types of Fc gamma-receptor

In our previous paper, we reported that guinea pig splenic lymphocytes expressed two distinct Fc-receptors for homologous IgG (Fc gamma Rs), one monospecific for IgG2 (Fc gamma 2R) and the other bispecific for IgG1 and IgG2 (Fc gamma 1/gamma 2R), when analyzed by EA-rosette assay. These Fc gamma Rs on the cells were further studied by using two monoclonal antibodies toward the Fc gamma Rs on guinea pig peritoneal macrophages (anti-Fc gamma 1/gamma 2R and anti-Fc gamma 2R antibody). The anti-Fc gamma 1/gamma 2R antibody completely inhibited the rosette formation of splenic lymphocytes with IgG1-sensitized sheep erythrocytes [EA(IgG1)]. On the other hand, EA(IgG2)-rosette formation was inhibited partially by anti-Fc gamma 2R but not by anti-Fc gamma 1/gamma 2R antibody. Complete inhibition of the EA (IgG2)-rosette formation was achieved by simultaneous additions of both anti-Fc gamma 2R and anti-Fc gamma 1/gamma 2R antibodies. The binding of IgG2 antibody complexed with ovalbumin to the cells was partially inhibited by either anti-Fc gamma R antibody, and complete inhibition occurred in the presence of both the antibodies, indicating that two types of Fc gamma R, Fc gamma 1/gamma 2R, and Fc gamma 2R, are expressed on the cells. The determination of these Fc gamma Rs on B and T lymphocytes by two-color flow cytometry showed that about 52% of B lymphocytes expressed Fc gamma 1/gamma 2R alone and 32% of the cells expressed both the Fc gamma Rs. On the other hand, about 12% of T lymphocytes was found to express Fc gamma 2R alone and the cells expressing Fc gamma 1/gamma 2R were in the minority (3.8%). T lymphocytes expressing both the Fc gamma Rs were not detected. These results show that guinea pig B lymphocytes bear two types of Fc gamma Rs and are heterogeneous with regard to their Fc gamma Rs and that T lymphocytes express Fc gamma 2R mainly.     

In vitro synthesis of human IgE by neonatal lymphocytes: enhancing effect of interferon-gamma

The present study demonstrates that neonatal human lymphocytes that are incapable of producing IgG and IgA antibodies may differentiate into IgE-secreting cells under the influence of IL4. Indeed, the addition of recombinant IL4 to cultures of unfractionated umbilical cord blood mononuclear cells (CBMC) induces a dose-dependent synthesis of IgE but not of the other classes of Ig. Moreover, IgE-secreting B lymphoblastoid cell lines can be derived from neonatal lymphocytes costimulated with EBV and IL4. Comparison of the mechanisms regulating the in vitro IgE synthesis by adult and neonatal lymphocytes indicates that in most cases IFN-gamma markedly potentiates the IgE synthesis in CBMC cultures whereas it has a reverse effect on adult lymphocytes. These reciprocal effects of IFN-gamma are specifically blocked by a neutralizing mAb to IFN-gamma; they are dose-dependent and they are observed when IFN-gamma is added at the initiation of the culture or shortly thereafter. Moreover, in a small number of cases IFN-gamma may also potentiate IgE synthesis by adult lymphocytes. The potentiation or the suppression of IgE synthesis by IFN-gamma is not explained by a differential effect of IFN-gamma on the production of soluble CD23 (sCD23); indeed in both cases IFN-gamma slightly increases the IL4-induced production of sCD23. Moreover, the spontaneous and the IL4-induced production of sCD23 by CBMC is comparable to that of adult lymphocytes. The IgE response is dependent upon the expression of Fc epsilon RII (CD23) inasmuch as it is specifically blocked by anti-CD23 mAb.     

Bacteria induce lymphokine synthesis polyclonally in human B lymphocytes.

We have studied the ability of various bacteria to stimulate human lymphocytes to produce leukocyte migration inhibitory factor (LIF). Mononuclear cells from adult and cord blood as well as purified T and B lymphocytes were stimulated with killed bacteria. The culture supernatants were tested for the presence of LIF by the agarose migration method. All nine bacterial strains tested activated unseparated mononuclear cells and B lymphocytes but not T cells to produce LIF. LIF was also present in cord blood cell cultures suggesting that the stimulation of lymphocytes was polyclonal rather than antigenic. Therefore, we propose that one of the physiologic functions of B lymphocyte lymphokines might be to form part of the nonspecific defense mechanisms against microbial invasion.     

Immune modulation of connective tissue functions: studies on the production of collagen synthesis inhibitory factor by populations of human peripheral blood mononuclear cells

It has been shown previously that a soluble factor(s) from human peripheral blood mononuclear cells was capable of specifically suppressing collagen synthesis by normal human dermal fibroblasts (S. A. Jimenez, W. McArthur and J. Rosenbloom, J. Exp. Med.150, 1421, 1979). In this communication, the cell sources and the conditions for synthesis of this collagen synthesis inhibitory factor (CSIF) are identified. CSIF production by mononuclear cells was directly related to the number of cells in culture and was significantly enhanced by a variety of mitogens and by antigens. Homologous serum or bovine serum albumin was required for CSIF production and maximal levels were reached 48 hr after stimulation. Thymus-derived lymphocytes appeared to be the main cells responsible for CSIF synthesis but B lymphocytes also produced the factor in response to proper B-cell mitogens. Preparations of plastic-adherent mononuclear cells were also found to produce increased CSIF but it was not possible to exclude completely the presence of T lymphocytes in these preparations and therefore, the cell source of CSIF in these preparations was not clearly established. Through the use of metabolic inhibitors it was shown that CSIF production required de novo protein synthesis but not cell division. Indo-methacin had no effect on either the production of CSIF or on CSIF-mediated inhibition of collagen synthesis. The results indicate that CSIF has the classic characteristics of a lymphokine and suggest a mechanism by which the immune response could modulate connective tissue function.     

Suppression of the late stages of mitogen-induced human B cell differentiation by FC gamma receptors (FC gamma R) released from polymorphonuclear neutrophils

During short incubation in serum-free medium, polymorphonuclear neutrophils (PMN) release soluble material that can be characterized as receptors for Fc IgG (Fc gamma R) on the following evidence: it agglutinates erythrocyte-IgG antibody (EAG) complexes, it prevents the binding of EAG to EAG-rosette-forming cells, and it binds to EAG-rosette-forming cells after modulation of their Fc gamma R, allowing the formation of 'passive' rosettes. These Fc gamma R were isolated by affinity chromatography on sepharose 4B-IgG. This material was shown to interfere with the differentiation of peripheral blood B cells into Ig-secreting cells in cultures stimulated by pokeweed (PWM) or Nocardia opaca (NOC) extracts. The number of Ig-secreting cells determined by a reverse hemolytic plaque assay using protein A-coated sheep erythrocytes was decreased by addition of Fc gamma R over a wide range of dilutions. The number of Ig-containing cells was diminished in PWM-stimulated cultures, but not in cultures stimulated with NOC. Fc gamma R did not affect cell viability, nor did they interfere with plaque-forming cell assay. Fc gamma R was not suppressive when added before the 3rd day or after the 6th day of culture. The suppressor factor was shown to remain associated with Fc gamma R after elution at acidic pH; it was removed by absorption on Sepharose 4B-IgG, but not on pepsin-digested F(ab')2 fragments. The suppressive factor as well as the capacity to restore EAG rosette formation by modulated lymphocytes were destroyed by heating (56 degrees C, 30 min) or by freezing and thawing. Properties of Fc gamma R released from PMN in this system are similar to those of Fc gamma R released from unstimulated human peripheral blood lymphocytes (PBL) and to those of mouse Ig-binding factor produced by alloactivated T cells or T cell lines.