A distinct "slow" cellular pathway involving soluble mediators for the T cell-instructed induction of monocyte tissue factor activity in an allogeneic immune response

Allogeneic stimulation of human lymphoid cells initiates a collaborative cellular pathway that relatively rapidly induces in monocytes the synthesis and cell surface expression of tissue factor, the initiating cofactor of the extrinsic coagulation pathway. T cells are required for the monocyte procoagulant generation, because the addition of autologous or allogeneic T cells fully reconstitutes the activity in otherwise nonresponding highly purified monocytes. Despite a strict T cell requirement, only low T cell to monocyte ratios are necessary for maximal PCA response. Our results further demonstrate that the collaborative signal from allogeneically stimulated T cells to effector monocytes is transferred by a soluble mediator rather than direct cell to cell contact. Other aspects of the present study include the observation that among normal peripheral blood lymphoid cells, monocytes elicit the strongest allogeneic PCA response. This response is clearly exceeded by that induced upon stimulation with Daudi lymphoblastoid B cells. Our data demonstrate the existence of a second distinct cellular pathway that mediates the lymphoid procoagulant response. This pathway differs from the previously characterized response to bacterial LPS in respect to: a) kinetics of T cell triggering; b) mediation by a soluble product; c) lack of genetic restriction of T cell; monocyte collaboration; and d) deficient capacity for direct T cell induction of the monocyte PCA response.     

Induction of monocyte procoagulant activity with OKT3 antibody

OKT3 monoclonal antibody (MoAb), a mouse MoAb against cluster of differentiation 3 (CD3) molecule, induced a large amount of procoagulant activity (PCA) in human peripheral blood mononuclear cells (PBM). The PCA-inducing capability in OKT3 MoAb was abolished by absorption with T lymphocytes or Sepharose-conjugated antibody to mouse IgG. Most of the PCA in PBM was associated with monocytes. There was a dose-dependent increase in PCA when increasing numbers of T cells were added to the monocytes in the presence of OKT3 MoAb. OKT3 MoAb did not induce PCA in either T cells or monocytes alone. T cells pulsed with OKT3 MoAb only in the presence of monocytes could induce PCA in monocytes. Culture supernatants (CS) from PBM stimulated with OKT3 MoAb did not enhance PCA in monocytes; however, it did induce PCA in the human monocyte-like cell line (U937) which differs in some properties from monocytes; this activity could be abolished by the MoAb against human interferon-gamma (IFN-gamma). Nevertheless, neither human IFN-gamma nor interleukin 1 or 2 had significant direct effect in inducing PCA in U937 cells; CS from either monocytes or T cells alone stimulated with OKT3 MoAb did not induce PCA in U937 cells. This apparent discrepancy suggests that there may be factors in CS that induce PCA in U937 cells only in the presence of IFN-gamma. The PCA induced in monocytes or U937 cells was tissue factor-like because of the dependence on coagulation factors V, VII, and X. These observations suggest that OKT3 MoAb is a potent T cell-dependent monocyte PCA inducer and stimulates T cells only in the presence of monocytes. The direct cellular interaction between monocytes and stimulated T cells appears to be necessary to elicit monocyte PCA with OKT3 MoAb stimulation. Thus, monocytes may play a dual role, not only as effector cells, but also as cells that collaborate with T cells after OKT3 MoAb stimulation so as to produce PCA.     

Endotoxin-induced T lymphocyte proliferation

The lymphocyte response to endotoxin (LPS) has been attributed largely to the action of this agent as a polyclonal activator of B lymphocytes. In this study we found that a cloned murine interleukin 2-dependent cytotoxic T cell line, CT 6, proliferates in response to LPS, thus providing the first evidence that T cells can be stimulated directly by LPS. The response was dose and time dependent and was blocked by polymyxin B, an inhibitor of LPS-induced mitogenesis. The fact that this is a cloned T cell line, free of other potentially contaminating lymphoid cell types, precludes the possibility that this proliferation is due to contaminating B lymphocytes or is mediated by macrophage-derived products such as interleukin 1. Moreover, highly purified splenic T lymphocyte populations (purified by negative/positive selection or by a rigorous column purification procedure) contain a small subpopulation (approximately 3%) of T cells that proliferate in response to LPS. This population is missing in the endotoxin-hyporesponsive C3H/HeJ mouse. As was observed in the CT 6 line, proliferation of splenic T cells in response to LPS was inhibited by polymyxin B. Furthermore, treatment of LPS-stimulated T cells with anti-T cell antibodies plus complement blocks the uptake of 3H-thymidine by these cultures. Exogenous interleukin 1 failed to stimulate the T cell cultures comparably to LPS and therefore cannot account for the degree of stimulation observed. These findings support and extend previous findings that suggested a role for an endotoxin-sensitive T cell population in the induction of certain responses, such as LPS-induced adjuvanticity of the lymphocyte-dependent LPS induction of macrophage procoagulant activity.     

Human T lymphocyte proliferation induced by a pan-T monoclonal antibody (anti-Leu 4): heterogeneity of response is a function of monocytes

Anti-Leu-4 is a murine monoclonal antibody that defines a molecule of 20,000 to 25,000 daltons present on all mature T lymphocytes in man. When cultured in the presence of 10 to 1000 ng/ml anti-Leu-4, the T cells of most individuals proliferate with peak responses on the third day of culture. T cells of both helper and suppressor lineages proliferate, but only in the presence of monocytes. Approximately 40% of individuals tested responded weakly or not at all to anti-Leu-4, despite normal responses to other stimuli. The variation in responsiveness between individuals could not be explained by differences in Leu-4 antigen density on the surface of T cells, differences in the rate of Leu-4 antigen modulation, or structural differences in the Leu-4 molecule as defined by the method of two-dimensional polyacrylamide gel electrophoresis. In the presence of monocytes from high responders, the T cells from low responders proliferated vigorously to anti-Leu-4, whereas monocytes from low responders failed to support proliferation by high responder T cells. On the other hand, low responder monocytes did not prevent T cells from proliferating in the presence of high responder monocytes. These results suggest that the failure of some individuals to respond to anti-Leu-4 is due to the absence or dysfunction of an essential monocyte population.     

Regeneration of T cell subpopulations after bone marrow transplantation: cytomegalovirus infection and lymphoid subset imbalance

Immune reconstitution was studied serially by using T lymphocyte cell surface differentiation antigens in 37 individuals receiving bone marrow transplants. Antigen expression was assessed by immunofluorescence analysis using monoclonal antibodies to T lymphocytes including Leu-3, which defines a T lymphocyte subpopulation in healthy individuals with T helper or inducer activity (L3+), and Leu-2, which defines a T lymphocyte subpopulation in healthy individuals with T helper or inducer activity (L3+), and Leu-2, which defines a T lymphocyte subpopulation with suppressor or cytotoxic activity (L2+). These studies demonstrated that the L2+ subpopulation regenerated more rapidly after transplant than did the L3+ subpopulation. Imbalances between these two T lymphocyte subpopulations, indicated by a decreased L3/L2 ratio, persisted for periods up to 12 mo post-transplant. Expression of a cell surface antigen associated with immature lymphocytes (OKT-10), and of HLA-DR (Ia-like) antigens was markedly increased during the post-transplant period. HLA-DR antigen expression did not appear related to immune activation in that increased reactivity was not detected with a monoclonal antibody (anti-TAC) specific for activated T cells. These observations in bone marrow transplant recipients and other disorders characterized by lymphoid restoration or immaturity indicate that inversion of the normal L3/L2 ratio and increased expression of OKT-10 and HLA-DR antigens may be features of a regenerating immune system. Furthermore, serial observation of individual patients indicated that infection with cytomegalovirus was associated with a progressive decrease in the L3/L2 ratio.     

Production of a human T lymphocyte chemotactic factor by T cell subpopulations

Concanavalin A-stimulated human peripheral blood mononuclear cells release a lymphocyte chemotactic factor. This lymphocyte chemotactic factor is produced optimally after 24 to 48 hr of culture and is not found before 3 hr of culture, which suggests that the factor is synthesized de novo and is not preformed and secreted after Con A stimulation. This is further supported by experiments showing that the protein synthesis inhibitors cycloheximide and puromycin totally prevent the production of the chemotactic factor. Experiments using cultured and uncultured T lymphocytes as responding cells show that cultured T cells respond more efficiently than uncultured T cells to this factor. Furthermore, the lymphocyte chemotactic factor preferentially stimulates T lymphocyte locomotion as compared to peripheral blood non-T lymphocyte migration. Fractionation of mononuclear cells into glass nonadherent lymphocytes, monocyte-enriched preparations, T lymphocytes, and non-T lymphocytes shows that lymphocyte chemotactic factor is produced by Con A-stimulated, glass nonadherent lymphocytes and T cells but not by monocytes or non-T lymphocytes. Further fractionation of T lymphocytes into Leu-2 and Leu-3 T cell subpopulations shows that the production of T lymphocyte chemotactic factor can be attributed to the Leu-2 suppressor/cytotoxic T cell subset. The generation of a T lymphocyte chemotactic factor by Leu-2 T cells may represent a means of recruiting other T cells to the site of its release.     

Alloantigen-specific cytotoxic and suppressor T lymphocytes are derived from phenotypically distinct precursors

Although Leu-2+ (OKT8+) T cells activated in the mixed lymphocyte reaction (MLR) mediate both alloantigen-specific cytotoxicity and suppression of alloantigen-induced proliferation, it is not known whether these functions derive from a single cell type or phenotypically distinct cells. This study was undertaken to examine the alloantigen-specific cytolytic and suppressor potential of two subpopulations of Leu-2+ cells distinguishable from one another on the basis of their binding to the monoclonal antibody 9.3. Leu-2+, 9.3+ and Leu-2+, 9.3- populations were purified from peripheral blood, cultured for 7 days with autologous helper/inducer (Leu-3+) cells and allogeneic non-T cells, and reisolated before testing for cytotoxicity and suppression. All detectable alloantigen-specific cytolytic activity was confined to the Leu-2+, 9.3+ subpopulation. Killing by this subset was specific for the HLA-A and B (class I) major histocompatibility complex (MHC) antigens of the priming cell. By contrast, suppression of proliferation was mediated predominantly by the Leu-2+, 9.3- cells, and suppression by this subpopulation was specific for the HLA-DR (class II) MHC antigens of the priming cell. The development of suppression by Leu-2+, 9.3- cells was unaffected by cyclosporin A (CsA), an agent shown previously to block the development of cytolytic but not suppressor cells in MLR. Alloactivated Leu-2+, 9.3+ cells were slightly inhibitory of fresh MLR, but this effect as well as the development of cytolytic cells was completely abrogated by CsA. These results indicate that suppressor and cytolytic Leu-2+ T cells activated in MLR are derived from distinct precursors separable by antibody 9.3.     

Autologous induction of the human T-cell-dependent monocyte procoagulant activity: a possible link between immunoregulatory phenomena and blood coagulation

Activated monocytes acquire the ability to induce clot formation in platelet-poor citrated human plasma. The generation of this procoagulant activity (PCA) is dependent upon an interactive pathway between monocytes and T lymphocytes. Here we show that an ongoing autologous mixed lymphocyte reaction (AMLR) can elicit a T-cell-instructed PCA. PCA was measured by the ability of the cells to accelerate the clotting time of pooled citrated platelet-poor human plasma. AMLR was measured by tritiated thymidine incorporation. PCA and AMLR had very similar kinetics. Correlation coefficients between both reactions ranged from 0.59 to 0.99. Addition of an anti-DR monoclonal antibody blocked both reactions. T-Lymphocyte-depleted cell populations did not increase their level of PCA after 6 days in culture. Addition of autologous T cells to the T-depleted population restored its ability to produce PCA. Cyclosporin A blocked the peripheral blood mononuclear cell ability to generate PCA. A lymphokine generated during the AMLR was able to induce PCA in normal mononuclear cells. The results indicate that self recognition activates monocytes to produce PCA and suggests that this mechanism may represent a link between immunoregulatory phenomena and blood coagulation.     

Cooperation between an anti-T cell (anti-CD28) monoclonal antibody and monocyte-produced IL-6 in the induction of T cell responsiveness to IL-2

CD28 is an Ag of 44-kDa Mr that is expressed on the membrane of the majority of human T cells and that is recognized by mAb 9.3. The functional effects of mAb 9.3 on peripheral blood T cells were studied. mAb 9.3 was not mitogenic, unless it was combined with PMA. When CD28 was cross-linked after binding of mAb 9.3 to the T cell by immobilized or soluble anti-mouse IgG, T cells proliferated in response to rIL-2, provided that monocytes were also present. The additional signal required for IL-2 responsiveness after cross-linking of CD28 could also be delivered in cultures of purified T cells by a cellfree monocyte culture supernatant. Expression of IL-2R on about 10% of the T cells was demonstrated by staining with an anti-IL-2R mAb, and was found to be largely restricted to CD4+ cells. The active compound responsible for the helper signal in the monocyte culture supernatant was identified as IL-6 because purified IL-6 (but not IL-1 beta) had similar activity and because an antiserum to IL-6 (but not an antiserum to IL-1 beta) neutralized the activity of the monocyte supernatant and blocked T cell proliferation. An anti-IL-2R antibody also completely inhibited T cell proliferation induced by the combination of mAb 9.3, IL-2, and IL-6. Our results provide evidence that cross-linking of CD28 induces functional IL-2R and that this activity is dependent on a helper signal provided by monocytes, more specifically IL-6. Moreover, our results indicate that IL-6 (previously called B cell stimulatory factor-2) is active on T cells. If a natural ligand for CD28 can be identified, the mechanism of induction of IL-2 responsiveness described here might explain how T cells become nonspecifically involved in an ongoing cellular immune reaction.     

Monocyte procoagulant inducing factor: a lymphokine involved in the T cell-instructed monocyte procoagulant response to antigen

A T cell-derived lymphokine couples the recognition of alloantigens by human T inducer cells to monocyte effector procoagulant activity (PCA). This collaborative cellular response results in expression of the functional tissue factor gene product and initiation of the extrinsic coagulation protease cascade as an inflammatory sequelae to the immune response. We now provide initial characterization of this lymphokine, provide evidence that it is a unique lymphokine, and designate it as monocyte procoagulant inducing factor (MPIF). MPIF was produced by alloantigen-stimulated, nylon wool-purified T cells and was not diminished by irradiation. MPIF active supernatants induced PCA in isolated monocytes, and the effect was not modified by the presence of T cells with the responding monocytes, consistent with a direct effect on the monocyte effector cells rather than an indirect effect via an intermediate accessory T cell. Full expression of PCA by monocytes was complete within 4 to 6 hours. MPIF activity in mixed lymphocyte culture (MLC) supernatants (MLC-SN) was stable at pH 2.0 for 24 hr, but was diminished after exposure to pH 10.5 for 30 min. MPIF activity was stable at 56 degrees C but was labile at 63 degrees C or higher. When characterized by chromatography on Sephadex G-100 superfine at either pH 7.2 or 3.6, the activity was recovered in a major discrete peak of about 55,000 daltons, and minor peaks of activity at about 14,000 and greater than 150,000 daltons. There was no correlation between the presence or concentration of INF-gamma, INF-alpha, IL 1, IL 2, GM-CSF, CSF-1, TNF-alpha, TNF-beta (lymphotoxin), or migration inhibitory factor (MIF) with MPIF activity. Each of the previously defined cytokines was analyzed directly for MPIF activity. INF-gamma, INF-alpha, GM-CSF, TNF-alpha, and CSF-1 did not possess MPIF activity over a wide range of concentrations. IL 1 and IL 2 lacked activity at concentrations present in MLC medium positive for MPIF; however, at higher concentrations each demonstrated slight activity. There was a poor correlation between MPIF and MIF activities in MLC-SN, and the content of MIF was insufficient to account for the expressed level of MPIF activity. The lack of identity of these cytokines with MPIF was supported by selected MLC medium that lacked IL 1, IL 2, and MIF and yet contained high MPIF activity. MPIF was additionally distinguished from IL 1, IL 2, and MIF on the basis of separation in Sephadex G-100 superfine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Monocyte-mediated augmentation of human natural killer cell activity: conditions, monocyte and effector cell characteristics

The characteristics of the effector cells and monocytes, and conditions required for the monocyte-mediated augmentation of human natural killer (NK) cell activity were investigated. Enriched null cell populations were further fractionated by Percoll centrifugation and used as effector cells. The LGL-enriched fraction was less susceptible than either the unfractionated cells or the other Percoll fractions to the monocyte augmentation when mixed with monocytes in the chromium-release assay and when precultured with monocytes for 12 hr, retrieved by carbonyl iron treatment, and tested for NK activity against K562. This differential susceptibility was reflected at the single cell level. The LGL-enriched Percoll fraction did not display the increase in target-binding cells with lytic activity that was exhibited by the other effector cell preparations after culture with monocytes. No differences in Leu-7 and Leu-11 phenotypes were detected between enriched null cells that had been cultured with and without monocytes for 12 hr. At the monocyte level, it was shown that pretreatment of the monocytes with LPS did not alter their NK-augmenting activity appreciably. Glutaraldehyde-fixed monocytes were not effective, and actinomycin D-treated monocytes were less effective than untreated or irradiated monocytes when mixed with enriched null cells in the assay. Actinomycin D-treated monocytes did not augment and possibly suppressed NK activity tested after 12-hr culture, and irradiated monocytes were less effective for augmenting NK activity than untreated cells. Monocyte-mediated augmentation could be detected when the medium used for null cell-monocyte coculture was supplemented with a) different lots of fetal bovine serum, b) human AB serum, c) autologous serum, or d) no serum. Polymyxin B and indomethacin did not alter the monocyte effect. Finally, the monocyte-mediated augmentation of human NK was not MHC restricted, since allogeneic combinations were also effective. These results suggest that 1) lymphocytes other than LGL participate in the monocyte-mediated augmentation of NK activity, 2) the augmentation is probably activational rather than maturational, 3) the monocytes must be viable to be effective when mixed with null cells during the assay, 4) de novo RNA and/or protein synthesis by the monocytes is required for the monocytes to induce augmented activity in null cells after 12-hr coculture, 5) prostaglandin synthesis and endotoxin are probably not involved in the augmentation, 6) the phenomenon is not MHC restricted, and 7) monocytes may express augmentative and suppressive activities concurrently.     

Two-color flow cytometry and functional analysis of lymphocytes cultured from human renal allografts: identification of a Leu-2+3+ subpopulation

The phenotype of T lymphocyte subsets present in renal biopsies showing acute cellular allograft rejection in six patients on cyclosporine have been characterized in situ by immunoperoxidase staining, and after expansion in vitro in interleukin 2 (IL-2) by two-color flow cytometry, sorting, and functional analysis. After 8 to 42 days in organ culture, both Leu-3+ (CD4) and Leu-2+ (CD8) subsets were found in each culture, in a ratio that varied from 0.2 to 5.0, which was not significantly different than the results of in situ immunoperoxidase staining of the uncultured biopsy. The cultured cells were almost all Leu-4+ (CD3) T cells (89% +/- 4), which expressed the activation markers DR (82% +/- 6) and the IL 2 (CD25) receptor (15% +/- 4). The Leu-3+ cells were largely Leu-8- (90% +/- 6), whereas a minority of the Leu-2+ cells were Leu-15+ (CD11) (26% +/- 4). Only a small fraction of the Leu-2+ cells stained for Leu-7 (8% +/- 6). Functional analysis of FACS-purified Leu-2-3+ and Leu-2+3- populations indicated that both subsets proliferated in response to graft donor antigens in a mixed lymphocyte reaction (MLR) and produced IL 2. Only the Leu-2+3- population demonstrated donor-specific cytotoxic activity. A minor subpopulation in each culture were both Leu-3+ and Leu-2+ (2.0%). Leu-2+3+ cells from one biopsy were purified to homogeneity (99.8%), and were found to express the T cell antigen receptor complex Ti/CD3 (WT-31+, Leu-4+), but not the common thymocyte antigen CD1 (OKT6). The Leu-2+3+ cells neither responded in the MLR, nor showed any cytotoxic capacity. The Leu-2+3+ cells were capable of IL 2 but not interferon-gamma production. None of the purified cultures demonstrated NK activity. A subset of the purified Leu-2+3+ cells lost Leu-2+ during 1 to 3 wk in culture, and became Leu-2-3+. These studies provide evidence that the cells that infiltrate renal allografts during rejection include alloproliferative, lymphokine-producing cells of both Leu-2+ and Leu-3+ subsets. The Leu-2+3- cells are also highly cytotoxic against donor lymphocytes, indicating the presence of helper independent cytotoxic T cells. A minor population of Leu-2+3+ T cells that do not express donor specific function was also identified.     

Lipopolysaccharide exposure during purification of human monocytes by adherence increases their recovery and cytolytic activity

Exposure of monocytes to lipopolysaccharide (LPS) during, but not after, adherence purification increased their cytolytic activity in short-term 51Cr-release assays against K562 target cells. In the absence of LPS only a minority of monocytes could be recovered by adherence. With 1 ng/ml to 10 micrograms/ml LPS present during the 1-hr adherence procedure, however, monocytes spread more extensively on serum-coated plastic and glass surfaces and virtually all of the monocytes in a mononuclear leukocyte preparation were recovered in the adherent fraction. While increasing the recovery of monocytes threefold, LPS exposure during adherence also increased monocyte purity as assessed by peroxidase staining, morphology, and indirect immunofluorescence with monoclonal Mo2. The proportion of Leu-11-positive NK cells in the adherent fraction did not change. Depletion of NK cells by treatment with anti-Leu-11b and complement eliminated cytolytic activity from the nonadherent, but not from the adherent, fraction isolated with LPS. Thus, addition of LPS during adherence produced a monocyte preparation with enhanced cytolytic activity not attributable to NK contaminants. To test whether LPS caused production of lymphokines that activate monocytes, we tested supernatants of unseparated mononuclear leukocytes for the capacity to stimulate purified monocytes for cytolysis. Such supernatants stimulated monocytes more effectively than LPS alone. We conclude that LPS stimulates monocytes for cytolysis most effectively during adherence purification because LPS allows the recovery of weakly adherent monocytes with high cytolytic capacity; also, LPS may stimulate production of lymphokines that further augment monocyte cytolytic activity.     

Immunoregulatory T cell circuits in man. Identification of a distinct T cell subpopulation of the helper/inducer lineage that amplifies the development of alloantigen-specific suppressor T cells

Regulation of the immune response in man is dependent on interactions between cells of helper/inducer (Leu-3+/T4+) lineage and cells of suppressor/cytotoxic (Leu-2+/T8+) lineage. By using the mixed leukocyte reaction (MLR) as a model system, we have shown previously that alloantigen-primed Leu-3+ cells induce autologous Leu-2+ cells to differentiate into suppressor T cells that specifically inhibit the response of fresh T cells to the original allogeneic stimulator cells. The current study was undertaken to analyze the roles in this suppressor circuit of subpopulations of Leu-3+ cells distinguished from one another on the basis of their binding or lack of binding to monoclonal anti-Leu-8 antibody. Although both Leu-3+,8- and Leu-3+,8+ T cells proliferated in allogeneic MLR, alloactivated Leu-3+,8+ cells alone induced proliferation and differentiation of Leu-2+ suppressor cells. Leu-3+,8+ cells also induced Leu-3+,8- cells to proliferate, and following their activation in this manner, such autoactivated Leu-3+,8- cells augmented the differentiation of Leu-2+ suppressor cells, but only in the presence of alloactivated Leu-3+,8+ cells. Furthermore, this effect, like the suppressor effect, was specific for the inducer cells, and thus indirectly for the HLA-DR antigens of the original allogeneic stimulator cells as well. These results indicate that alloantigen-primed Leu-3+,8+ cells not only activate specific Leu-2+ suppressor cells but also activate specific Leu-3+,8- suppressor-amplifier cells, and in combination, these cells exert potent feedback inhibition of MLR.     

Recombinant IFN-gamma synergizes with lipopolysaccharide to induce macrophage membrane procoagulants

Fibrin deposition is an important histopathologic feature of inflammation and is mediated, in part, by monocyte/macrophage procoagulants. rIFN gamma acted in synergy with suboptimal levels of bacterial LPS by priming thioglycollate-induced mouse peritoneal exudate cells (TG-PEC) to express high levels of surface procoagulant. TFN-alpha beta, TFN-alpha, IL-1, either alone or in combination with LPS or IFN-gamma, had no effect on macrophage procoagulant activity expression. In contrast to the dramatic increases of macrophage procoagulant activity induced by IFN-gamma/LPS, on exudate macrophages, normal peritoneal macrophages, or peripheral blood monocytes were unresponsive suggesting that the state of activation of the macrophage determines reactivity. IFN-gamma induced a Factor VIIa-like activity detected only after cell disruption. Synergy between LPS and IFN-gamma-induced procoagulants may occur as the result of the assembly of the thromboplastin (induced by LPS), Factor VII/VIIa complex on the macrophage surface. RNA synthesis was required for procoagulant induction. Procoagulant expression may, as for other cytokines involved in inflammatory responses, be regulated by short lived repressor proteins as low dose cycloheximide superinduced procoagulant responses to both LPS and IFN-gamma and caused the extracellular expression of procoagulant in response to IFN-gamma. This study suggests an important role for IFN-gamma in the assembly of components of the extrinsic coagulant cascade on the macrophage surface. The synergy between IFN-gamma and LPS may moderate macrophage-initiated fibrin deposition characteristic of inflammatory responses.     

Two phenotypically distinct suppressor T cell subpopulations inhibit the induction of B cell differentiation by phytohemagglutinin

Human B lymphocytes can be induced to differentiate into antibody-secreting plasma cells by Leu-3+ T lymphocytes stimulated with pokeweed mitogen (PWM), a polyclonal T cell activator. In contrast, other polyclonal T cell mitogens, such as phytohemagglutinin (PHA), also activate Leu-3+ T cells but are relatively ineffective inducers of B cell differentiation. We have performed a series of experiments to investigate the mechanism underlying this apparent paradox. When human B cells were cultured with unfractionated T cells and PWM or PHA, only PWM was able to induce plasma cell formation and immunoglobulin (Ig) secretion. However, when the T cells were treated with mitomycin C (MMC) before culture, both PWM and PHA were able to induce significant B cell differentiation. These data indicated that both mitogens were able to activate the helper T cells required for B lymphocyte differentiation and suggested that MMC-sensitive suppressor T cells were responsible for inhibiting the induction of antibody-secreting cells by MMC-untreated T cells stimulated with PHA. Phenotypic analysis of the T cells capable of suppressing PHA-induced B cell differentiation revealed that small numbers of either Leu-2+ or Leu-3+ T cells could profoundly suppress the B cell differentiation induced by PHA. In contrast, significant suppression of PWM-stimulated B cell differentiation was observed only with relatively large numbers of Leu-2+ T cells. These data confirm previous reports that OKT4+/Leu-3+ T cells can suppress human B cell differentiation and indicate that the difference in B cell differentiation induced by PWM and PHA with MMC-untreated T cells is largely a reflection of the relative potency of these mitogens to activate these phenotypically distinct suppressor T cell subpopulations.     

Inhibition of endotoxin-induced monocytic procoagulant activity by n-alcohols and anesthetics.

1. The effect of n-alcohols (methanol and ethanol) and anesthetics (lidocaine, thiopental, methohexital and thiamylal) on procoagulant activity (PCA) in human peripheral-blood monocytes and non-adherent cultured leukemia promonocytic U937 and THP-1 cells was examined herein. 2. Exposure of whole blood to ethanol showed no effect on PCA in human monocytes. However, ethanol dose-dependently inhibited LPS-induced PCA in isolated human monocytes. 3. In THP-1 cells, ethanol had no significant effect on PCA in either non-challenged or LPS-induced status. However, the induction of PCA by LPS was substantially inhibited when cells were pretreated with 1% ethanol (v/v) for 72 hr. 4. In U937 cells, n-alcohols and anesthetics resulted in dose-dependent depressions in PCA. Importantly, the percent reduction in LPS-induced PCA was much more pronounced than that in non-challenged PCA. 5. These data clearly suggest that n-alcohols and anesthetics readily inhibit the LPS-stimulatory action on monocytic PCA.     

Synergistic activation of human monocytes by granulocyte-macrophage colony-stimulating factor and IFN-gamma. Increased TNF-alpha but not IL-1 activity

TNF-alpha and IL-1 activities and PGE2 levels were investigated in the supernatants of highly purified human monocytes cultured for 18 h with recombinant human granulocyte-macrophage CSF (GM-CSF). GM-CSF alone did not stimulate IL-1 or TNF-alpha activities or the production of PGE2. GM-CSF with IFN-gamma, but not with LPS, consistently activated the monocytes for TNF-alpha activity. In contrast, for increased IL-1 activity, GM-CSF synergized weakly and irregularly with LPS, but not at all with IFN-gamma. For the third monocyte product investigated, GM-CSF was a weak and inconsistent inducer of PGE2 and only in the co-presence of IFN-gamma. Thus, GM-CSF can elicit different responses in human monocytes depending both on the co-stimulus as well as the monocyte product being investigated.     

Colony formation by subpopulations of human T lymphocytes. III. Antigenic phenotype and function of colony-forming cells, colony cells, and their expanded progeny

The antigenic phenotype of individual PHA-induced T lymphocyte colonies was studied with a direct immunofluorescence technique using fluorescein-labeled anti-Leu-2a and anti-Leu-3a antibodies. Of the colonies grown from mononuclear peripheral blood cells 85% were Leu-3a+ (inducer/helper phenotype), 12% were Leu-2a+ (suppressor/cytotoxic phenotype), and 3% contained equal numbers of Leu-2a+ and Leu-3a+ cells. Fluorescence-activated cell sorter (FACS) separated T-cell subsets showed that Leu-2a+ cells and Leu-3a+ cells form exclusively Leu-2a+ and Leu-3a+ colonies, respectively. Leu-3a+ cells formed colonies in both the absence and presence of conditioned medium (PHA-CM), whereas colony formation by Leu-2a+ cells was absolutely dependent on PHA-CM. Mixing experiments with FACS-separated T-cell subsets showed that Leu-2a+ cells inhibit colony formation by Leu-3a+ cells in a cell dose-dependent manner both in the presence and absence of PHA-CM. Phenotype analysis of individual colonies from mixing experiments strongly suggested monoclonal proliferation in the present colony assay system. The majority of expanded T-cell colonies showed helper activity in a reverse hemolytic plaque-forming B-cell assay, although to a lesser degree as compared to that of freshly isolated T lymphocytes.     

Activation of human B lymphocytes. IX. Modulation of antibody production by products of activated macrophages.

Human monocytes, after in vitro activation by mixed lymphocyte culture (MLC) supernatants produce a monokine (MK) that enhances the plaque-forming cell (PFC) response of pokeweed mitogen (PWM)-stimulated human B lymphocytes. Technical conditions and kinetics of MK production were established. Irradiation of monocytes (5000 rads) does not abolish MK production but heat-killed cells are unable to release the factor. Highly T cell-depleted monocyte populations still produced the PFC-enhancing factor. The same MK has an inconsistent enhancing effect on the PFC responses of lipopolysaccharide (LPS) and nocardia water-soluble mitogen (NWSM)-stimulated B cells. Other macrophage activators such as LPS, phytohemagglutinin (PHA), and latex particles failed to induce consistently the liberation of the PFC-enhancing MK. The target cell for the MK activity on PWM-stimulated B cells appears to be the B lymphocyte itself. These studies demonstrate that soluble monocyte products can have substantial modulatory effects on human B cell function.