The Fc receptor on thymus-derived lymphocytes. I. Detection of a subpopulation of murine T lymphocytes bearing the Fc receptor.

Utilizing the fluorescence-activated cell sorter (FACS) and washed murine antibody-antigen complexes formed in antibody excess, we have demonstrated the presence of the Fc receptor on the surface of a distinct subpopulation of murine T lymphocytes. No differences in intensity of labeling with the complexes was observed when the Fc+ T lymphocytes were compared with Fc+ B lymphocytes. The majority of Fc+ T lymphocytes are small lymphocytes determined by light-scattering characteristics on the FACS. Separating Fc+ from Fc- T lymphocytes from spleens of mice primed 1 wk or 1 mo previously with keyhole limpet hemocyanin (KLH) revealed that the T cells capable of cooperating with DNP-KLH primed B cells to give an adoptive anti-DNP PFC response do not bear the Fc receptor.

The Fc receptor on thymus-derived lymphocytes: II. Mitogen responsiveness of T lymphocytes bearing the Fc receptor.

The responsiveness of purified Fc- and Fc+ T lymphocytes, isolated from normal spleen cell populations by cell sorting on the fluorescence activated cell sorter, has been examined. Although both Fc- and Fc+ T cells responded to phytohemagglutinin, the response to concanavalin A (Con A) was found to be a characteristic of the Fc+ T lymphocyte. The poor responsiveness of the Fc- T cells to Con A was shown not to be due to a requirement of either different concentrations of Con A or for adherent cells. The addition of Fc+ T cells to the Fc- T cells in a ratio of 1:3 resulted in a mitotic response not significantly different from that observed with the purified Fc+ T cells alone and up to 15-fold greater than that of Fc- T cells alone. It is suggested that the Fc T cells can be recruited into mitosis as a result of Con A stimulation of the Fc+ T cells.

Regulation of the immune system by synthetic polynucleotides. V. Effect on cell-associated immunoglobulin receptors and immunological memory.

Addition of polyadenylic-polyuridylic acid in complex form (poly A:U) without antigen to a suspension of spleen cells obtained from BALB/Aj mice primed 6 wk previously with human gamma-globulin (HGG) resulted in an immediate fourfold increase over background number of anti-HGG rosette-forming cells (RFC). Culture of similar cells in the presence of puromycin for 1-6 hr before poly A:U did not significantly reduce the response. Continued culture of primed spleen cells in the presence of poly A:U, resulted in a decrease of RFC to background levels within an hour followed by an increase again 6 hr later. This later increase in RFC was inhibited by addition of puromycin to the culture medium. The nonspecific stimulation by poly A:U of antibody production by primed spleen cells also was induced in vivo. Increases in splenic RFC were detectable 6 hr after intravenous injection of poly A:U alone, without antigen, into primed mice. The response peaked at 18 hr and had dissipated completely within 3 days. A second injection of poly A:U 24 hr or later after the first injection resulted in a second response, similar to the first with respect to kinetics and intensity. Rosette formation by poly A:U-stimulated cells could not be inhibited by mitotic poisons, but was inhibited by treatment of the cells with goat anti-mouse gamma-globulin serum, suggesting that the antibody involved was a 7S gamma-globulin. The decrease in RFC induced by culture of primed cells for 1 hr in poly A:U paralleled a decrease in secondary responsiveness of the cells to antigen. This poly A:U-induced inhibition of secondary responsiveness could be reversed by suspending the treated cells in supernatant fluids derived from poly A:U-stimulated cultures. The reversal was specific in that supernatant fluids removed from bovine serum albumin (BSA)-primed cells by poly A:U did not stimulate the response of HGG-primed cells to HGG. However supernatant fluids from BSA-primed cells caused the production of anti-HGG RFC if BSA rather than HGG was used as triggering antigen. The active factor in the supernatant fluids appeared to be a 7S gamma-globulin since activity was lost after 45 min incubation of the supernatant fluids in the presence of goat anti-mouse 7S gamma-globulin serum.

Studies on membrane-bound receptors for antigen. Preparation of populations of receptor-depleted lymphocytes.

THE EFFECT OF POLYADENYLIC: polyundylic acid complexes (poly A:U) on the amount of antibody on the surface of various populations of mouse lymphoid cells has been investigated by means of a sensitive measure of such activity-the binding by primed cell populations of beta-galactosidase (betaGZ) as an antigen. The sensitivity derives from the liberation of fluorescein from an artificial substrate, fluorescein-di-beta-galactopyranoside (FDbetaG). After incubation with 100 ng/ml of poly A:U, only 40% of the cells previously showing antigen-binding were still active. The optimum range of activity lay between 0.01-1.0 microg/ml poly A:U. Such cells showed increased RNA and protein synthesis as indicated by [(3)H]uridine and [(14)C]amino acid incorporation. The polynucleotide effect was abolished by incubation of the cells with sodium azide or iodoacetate, but not by puromycin. When the proteins on the cell surface were labeled by (125)I, poly A:U caused their release into the medium. Reports by others that the enhancing effect of polynucleotides on the immune response involves the adenylcyclase system are consistent with the finding reported here that reduction of binding by dibutryl 5'-cyclic monophosphoric acid (cAMP) and poly A:U were parallel in extent, and that theophylline and poly A:U acted synergistically in suboptimal concentrations of each.

The Fc receptor on thymus-derived lymphocytes. III. Mixed lymphocyte reactivity and cell-mediated lympholytic activity of Fc- and Fc+ T lymphocytes.

The involvement of Fc- and Fc+ T cells, separated on the fluorescence-activated cell sorter, in proliferative and cytotoxic responses to alloantigens was examined. The cytotoxic lymphocytes generated by in vivo exposure to allogeneic tumor cells were shown to express the Fc receptor. The proliferative responses to alloantigen exposure in mixed lymphocyte cultures was equivalent in intensity for unseparated T cells, the Fc+ T-cell fraction, and the Fc- T-cell fraction isolated from nonsensitized spleen cells. In contrast, the cytotoxic responses generated by the Fc- T-cell fraction (less than 1% Fc+) were much weaker than the cytotoxic responses generated by the Fc+ T-cell fraction (80-90% Fc+), and the responses of the Fc+ T-cell fraction were generally weaker than, or equal to the responses of unseparated T cells (Fc- T less than Fc+ T less than or equal to unseparated T). Mixtures of the Fc- and Fc+ T-cell fractions mounted stronger cytotoxic responses than the sum of the responses of either fraction alone. Examination of the Ly phenotypes of the synergizing populations revealed that the CL precursor activity (Ly-2+ T cells) resided in the Fc- T-cell population, and that the amplifier T-cell activity (Ly-1+ T cells) resided in the Fc+ T-cell population. The data are discussed in terms of T-cell heterogeneity, differentiation, and intercellular interaction.

The Fc receptor on thymus-derived lymphocytes. IV. Inhibition of binding of antigen-antibody complexes to Fc receptor-positive T cells by anti-Ia sera.

Treatment of splenic T lymphocytes with anti-Ia antiserum inhibits the binding of antigen-antibody (AgAb) complexes to the majority (less than 50%) of Fc receptor-positive (FcR+) T cells. A similar inhibition was observed with anti-H-2D and anti-H-2K sera but not with anti-Thy 1.2. Despite the presence of Ia determinants on peripheral T cells, as established by the inhibition of AgAb binding, Ia could not be detected on peripheral T cells by immunofluorescence assays. Data obtained with the AgAb-binding inhibition assay indicate that determinants controlled by loci mapping in the I-A and I-C, S, or G regions are present on the FcR+ T cells. Evidence is presented that subpopulations of T cells within the FcR+ T-cell population may be distinguishable on the basis of which I-region-controlled determinant is expressed. The data are discussed in terms of phenotypic and functional heterogeneity of T lymphocytes.

Macrophage activation by T cells: cognate and non-cognate signals.

Both tumor necrosis factor-alpha and interferon-gamma are involved in the activation of macrophage cytocidal/cytostatic effector function. Recent studies provide evidence that, in non-septic inflammatory disease, T cells may activate macrophages primed by interferon-gamma either by providing tumor necrosis factor-alpha (in soluble or membrane-anchored form) or by inducing macrophage tumor necrosis factor-alpha production by antigen-non-specific cognate interactions. Conversely, T cells may inhibit macrophage activation by producing cytokines that inhibit either tumor necrosis factor-alpha production or interferon-gamma receptor signaling.

T cell rescue of monocytes from apoptosis: role of the CD40-CD40L interaction and requirement for CD40-mediated induction of protein tyrosine kinase activity.

Circulating monocytes have a limited life span and will undergo apoptosis in the absence of specific stimuli. Recent studies have demonstrated that monocytes can be rescued from apoptosis via lipopolysaccharide (LPS) activation or stimulation with interleukin-1 or tumor necrosis factor-alpha. Based on previous studies from our laboratory, we hypothesized that, in nonseptic (e.g., autoimmune) inflammation, the presence of activated T cells may enhance monocyte longevity through T cell contact-dependent signaling. Plasma membranes prepared from 6 h activated (TmA) and resting (TmR) purified CD4+ T cells were added to resting elutriation-purified monocytes cultured in serum-free medium. Cells were assayed for degree of apoptosis occurring over a 72-h incubation using both agarose gel electrophoresis and flow cytometry. The addition of TmA (but not TmR) was capable of blocking monocyte apoptosis and the ability of TmA to rescue monocytes was abrogated by the addition of anti-CD40L antibodies. Rescue of monocytes from apoptosis could also be mediated by direct cross-linking of monocyte CD40. Inhibitors of tyrosine kinase activity blocked both TmA and anti-CD40-mediated rescue of monocytes from apoptosis, suggesting a primary role of a tyrosine kinase signaling pathway in the events controlling monocyte longevity.

Cytokine priming reduces dependence on TNF-R2 for TNF-alpha-mediated induction of macrophage nitric oxide generation.

In the presence of interferon-gamma (IFN-gamma), human tumor necrosis factor-alpha (Hu-TNF-alpha), which binds to murine TNF-alpha receptor type 1 (TNF-R1) but not to murine TNF-R2, was effective in inducing nitric oxide (NO) production in spleen-derived macrophages (M phi), albeit at concentrations 12.5-fold greater than those required by murine TNF-alpha (Mu-TNF-alpha), to achieve the same result. Addition of anti-TNF-R1 completely inhibited the Mu-TNF-alpha-mediated induction of NO, demonstrating that TNF-R1 is critical to the IFN-gamma-dependent TNF-alpha-mediated induction of M phi effector function. However, treatment with anti-TNF-R2 resulted in a partial inhibition of M phi activation. Spleen-derived M phi were more dependent on TNF-R2 than RAW 264.7 or peritoneal M phi based on their responsiveness to Hu-TNF-alpha. Priming of spleen-derived M phi with either IFN-gamma or granulocyte-macrophage colony-stimulating factor (GM-CSF) heightened the maximal responses to both TNF-alpha species and increased the overall effectiveness of Hu-TNF-alpha without increasing expression of either TNF-alpha receptor. The dependence of spleen-derived M phi on both TNF-alpha receptors for signaling the induction of effector function supports an active signaling role for TNF-R2 in its synergy with TNF-R1 rather than a passive ligand passing role.

T cell signaling of macrophage function in inflammatory disease.

Macrophages play diverse roles in episodic T cell-mediated inflammatory diseases such as multiple sclerosis and rheumatoid arthritis, function as accessory cells for T cell activation, as pro-inflammatory cells, as effector cells which mediate tissue damage, and as anti-inflammatory cells which promote wound healing. In addition to the many roles of T cell-derived cytokines in differentially modulating these diverse macrophage activities, research over the last few years has demonstrated that contact-dependent signaling which occurs during T cell-macrophage adhesion is a critical triggering event in the activation of macrophage function. Substantial research emphasis has been placed on CD40 as a mediator of contact dependent signaling. However, other membrane-anchored receptor:ligand pairs may also contribute to the stimulation of macrophage function. This is a brief review of the rapidly expanding, but still incomplete, knowledge of how T cells, through both contact-dependent and cytokine signals, regulate macrophage function during inflammatory disease.

A requirement for native (undenatured) IgG for detection of species specificity of the IgG-Fc receptor on lymphocytes.

The species specificity of the Fc receptor involved in binding of preformed antibody-antigen complexes was examined using complexes of deaggregated 7S antibodies and albumin antigens. Complexes prepared with mouse antibodies bound to 3-4 times as many cells as complexes prepared with rabbit, guinea pig, or goat antibodies. Using lymphocytes from each species, it was shown that homologous complexes consistently labeled a higher frequency of cells than heterologous complexes. The binding of heterologous complexes seemed to be generally restricted to monocytic populations which were also capable of binding monomeric 7S immunoglobulins. It is concluded that the lymphocytic Fc receptor for antigen-complexed 7S immunoglobulin displays strict species specificity. Heat aggregation of the heterologous immunoglobulins eliminated the species restriction on their binding to the lymphocytic Fc receptor. It is therefore suggested that artificially aggregated immunoglobulins may not be reliable probes for the specificity of the Fc receptors.

Immunosuppressive therapy versus bone marrow transplantation for children with aplastic anemia.

A total of 15 patients 1 to 16 years of age were treated for aplastic anemia (13 of a severe degree) and followed-up for a mean of 24 months (range 2 to 64 months). Six patients had an HLA-matched sibling and underwent allogeneic bone marrow transplantation. Nine patients who lacked a suitable donor were given immunosuppressive therapy. Antithymocyte globulin was the initial treatment for eight of these nine patients. Two patients who failed to respond to antithymocyte globulin were then treated with cyclosporine A. Pretreatment age, hematologic measurements, duration of follow-up, and interval prior to therapy were similar between the two groups. All of the patients receiving bone marrow transplants had a complete response and now have normal blood cell counts. Six of nine patients (67%) responded to antithymocyte globulin and are now transfusion free, although three have mild thrombocytopenia. Both patients given cyclosporine A had a good response and are also transfusion free. Patients who underwent marrow transplantation had a significantly shorter period of transfusion dependence for RBCs (9 v 4 weeks, P less than .005) and platelets (5 v 21 weeks, P less than .05). The patients given immunosuppressive therapy have significantly lesser platelet counts in follow-up but have similar values for both hemoglobin and absolute granulocyte counts. Although HLA-matched bone marrow transplantation leads to a faster and more complete recovery for children with aplastic anemia, immunosuppressive therapy can provide a good outcome for children with this disorder.

Panning T cells on vascular endothelial cell monolayers: a rapid method for enriching naive T cells.

A key functional/phenotypic difference between naive and memory T cells is the ability of memory and activated T cells to home to sites of inflammation by adhering to vascular endothelial cells. To determine if this trait could be used to separate naive T cells from memory T cells, CD4+ T cells were incubated with monolayers of IFN-gamma-primed vascular endothelial cells after which the phenotypic and functional characteristics of the nonadherent population were assayed. The nonadherent population 1) contained a five-fold decrease in the frequency of cells displaying the CD44(high)/CD45RB(low) "memory" phenotype and 2) responded well to allostimulation but displayed a reduced ability to respond to immobilized anti-CD3 antibody and, when isolated from ovalbumin-immunized mice, displayed a reduced recall response to ovalbumin in vitro. These studies demonstrate that rwo brief incubations of T cells with monolayers of IFN-gamma-primed endothelial cells can significantly enrich for naive T cells as determined by both phenotypic and functional analyses.

The presence of a receptor for complement on T lymphocytes. Restriction of the complement receptor to Fc-receptor-bearing T lymphocytes.

Using rhodaminated guinea pig anti-ovalbumin:ovalbumin:complement complexes, a fluoresceinated monoclonal anti-Thy 1 antibody and a FACS-II equipped with dual fluorescence detection channels, we find that 25-30% of Thy 1+ splenocytes express a receptor for complement. That a receptor for complement is involved in binding the guinea-pig AgAb:C' complexes is supported by the observations that: a) guinea-pig complexes, which were not treated with a serum source of complement or which were treated with either fresh serum in the presence of EDTA or with heat-inactivated serum, do not bind to the T lymphocytes, and b) heat-aggregated human gamma globulin, which effectively inhibits binding of mouse AgAb complexes to the aFcR gamma, has no effect on the binding of guinea-pig AgAb:C' complexes to the T lymphocytes. By analyzing cell subpopulations isolated by cell sorting, it is demonstrated that the C'R-positive T lymphocyte clearly delineates a major subpopulation of aFcR gamma-positive T lymphocytes, whereas no cells are found bearing a C'R, while lacking the aFcR gamma. The implications of the presence of a C'R in immunoregulation are discussed.

LPS stimulation of TNF-receptor deficient macrophages: a differential role for TNF-alpha autocrine signaling in the induction of cytokine and nitric oxide production.

To evaluate the role of autocrine TNF-alpha signaling in macrophage activation, immortalized macrophages from normal mice (B6/J2) and from mice containing gene targeted disruptions of the type 1 and type 2 TNF-receptor genes (TRN) were stimulated under CD14-dependent or serum-free conditions. Although the B6/J2 and TRN clones mounted similar nitric oxide responses to LPS in the presence of serum, the TRN macrophages responded poorly when stimulated with LPS under serum free conditions. LPS stimulation of TRN and B6/J2 under serum-free conditions resulted in equivalent levels of IL-1beta, TNF-alpha, and iNOS gene expression. However, Western blot analysis revealed that iNOS protein production by TRN was 2-fold lower than that produced by B6/J2. These results indicate that autocrine TNF-alpha stimulation contributes to the signaling pathways initiated by ligation of LPS receptors in the absence of LBP and is involved in iNOS post-transcriptional regulation.

Cytostatic activity of in vitro generated macrophages: evidence for a prostaglandin-independent reversible cytostatic mechanism.

Culture of spleen cells for 5 days has previously been shown to result in the generation of strongly adherent cells from nonadherent precursors. In the current report it is shown that the majority (85-95%) of adherent cells are Mac-1+, FcR+, Thy 1.2- macrophages. Expression of effector activity by these macrophages requires exposure to activating signals. Coculture of the macrophages with Con A-stimulated spleen cells results in the expression of cytostatic activity against lymphocytic and monocytic tumor cell lines. Significant cytostatic activity is apparent within 6 hr after addition of the activating cells. Culture supernates of Con A-stimulated spleen cells (CAS-CM) are not effective in inducing cytostatic activity in the adherent macrophage population either alone or in the presence of additional Con A. However, stimulation of the culture generated macrophages with LPS in the presence of CAS-CM does induce cytostatic activity. The effector cell must be metabolically active in order to effect cytostasis insofar as heat fixation of the culture generated macrophage population eliminates effector activity. Proliferation of the tumor cells is significantly reduced after a 4-hr incubation period with the activated macrophages and is reduced two- to threefold after an 8- to 12-hr incubation period. The cytostatic effect is rapidly reversible. Proliferative activity of the tumor cells returned to control level within 12-24 hr after removal from activated macrophages. Cell cycle analysis indicated that the target cells were not arrested in a single stage of cell cycle, although an increase in frequency of cells in G1-phase was observed. Fluorescence analysis of bromodeoxyuridine (BrdU) incorporation rate demonstrated that the rate of DNA synthesis was reduced in all of the cells in the target population and that the mean rate of BrdU incorporation of the inhibited cells was three- to fivefold lower than control cells. RNA and protein synthesis were not affected to the same degree as DNA synthesis. The cytostatic effect was not mediated by prostaglandins or thymidine insofar as addition of indomethacin and 2-deoxycytidine did not prevent the cytostatic activity of the macrophages. The supernates of activated macrophages contained little inhibitory activity especially when indomethacin was included in the culture medium (19% inhibition of tumor cell proliferation by 1:1 dilution of supernate). The activity that was present could be eliminated by dialysis against fresh culture medium using Spectropor membranes with a 1000-Da molecular cutoff.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of IL 2 responsiveness of mitogen-stimulated lymphocytes by SMLR-generated plastic adherent suppressor cells.

The proliferative response of spleen cells to concanavalin A (Con A) can be abrogated by plastic adherent suppressors generated in syngeneic mixed lymphocyte cultures of spleen cells. The addition of exogenously produced interleukins does not overcome the suppression, indicating that the suppressors are not simply competing for available growth factors. To examine the effect of the suppressors on IL 2 production and responsiveness, spleen cells were stimulated with Con A in the presence of the suppressors and assayed both for ability to produce IL 2 and for ability to bind IL 2. The culture supernates of suppressed spleen cells contained normal titers of biologically active IL 2, indicating that the suppressors do not inhibit IL 2 production or inactivate IL 2 after it is produced. Since IL 1 is required for IL 2 production, the failure of the suppressors to affect IL 2 production suggests that the suppressors have no effect on either production of, or responsiveness to, IL 1. The suppressed cells did, however, display a substantial reduction in ability to absorb IL 2 activity from a standard IL 2 preparation. The reduced frequency of IL 2 reactive cells did not appear to be due to a cytotoxic reaction insofar as no difference in viable cell recovery was noted between suppressed and nonsuppressed cultures at the time of assay. The suppressors therefore appear to inhibit the expression of IL 2 responsiveness without inhibiting the production of IL 2.

Antigen-specific activation of effector macrophages by IFN-gamma producing (TH1) T cell clones. Failure of IL-4-producing (TH2) T cell clones to activate effector function in macrophages.

IFN-gamma-producing (TH1) and IL-4-producing (TH2) clones were assayed for their ability to directly induce cytostatic activity in macrophages generated from splenic myeloid precursors (M phi-c). In the presence, but not in the absence, of antigen, TH1 clones activated the M phi-c to inhibit the growth of P815 tumor cells in vitro. TH2 clones were not able to activate such effector activity in the M phi-c. The M phi-c did effectively present Ag to the TH2 clones as evidenced by the proliferation of TH2 cells cultured with Ag in the presence, but not in the absence, of M phi-c. Therefore, although both TH1 and TH2 were activated by cognate interaction with antigen presenting M phi-c, only TH1:M phi-c interactions displayed reciprocity resulting in activation of the M phi-c. TH1-derived lymphokines or rIFN-gamma, in the presence of LPS, could activate proteose-peptone elicited M phi, resident peritoneal M phi, and M phi-c whereas neither TH2-derived lymphokines nor rIL4 could induce detectable activity in any of the 3 M phi populations. IFN-gamma, in the absence of LPS, could activate the elicited M phi and to a lesser and more variable degree, the resident M phi Only the M phi-c consistently required both IFN-gamma and LPS for induction of cytostatic activity. Since M phi-c consistently required at least two signals for activation, the ability of TH1-derived lymphokines to synergize with TH2 cells in M phi activation was examined. TH2 could activate the Ag-presenting M phi-c in the presence of IFN-gamma. The ability of added IFN-gamma to synergize with TH2 indicates that the cognate interaction between TH2 and antigen presenting M phi-c does result in delivery of at least one of the signal required for M phi activation.

Suppression of lymphocyte proliferative responses: demonstration of two stages occurring in the in vitro generation of suppressor macrophages.

The suppressor macrophages generated by in vitro culture of spleen cells are shown to be derived from nonadherent splenic precursors. The induction of suppressor macrophage generation requires the presence of both plastic-adherent and plastic-nonadherent spleen cells during the first 24 to 48 hr of culture. After this induction period, the primed nonadherent cells can continue to generate suppressor macrophages through several serial transfers. The primed nonadherent spleen cell population is predominantly comprised of cFcR+ cells (40 to 50%) and Thy-1+ cells (40%), many of which appear to be undergoing blastogenesis. The generation of suppressor macrophages appears to proceed in two stages. The first stage is radiosensitive and has a minimum duration of 1 to 2 days. Although this stage initially coincides with the inductive period, it is also required for the continuous propagation of the suppressor macrophages upon serial transfer of the nonadherent spleen cells. The second stage is radioresistant and has a maximum duration of 2 to 3 days. The in vitro events thus bear similarities to events occurring during in vivo differentiation and activation of effector macrophages. It is suggested that the in vitro generation of suppressor macrophages may be one component of syngeneic mixed lymphocyte responses when spleen cells are used as the responding population, and that the suppressors represent part of a regulatory system controlling lymphocyte and macrophage differentiation and activation.