Activated T lymphocytes regulate hyaluronan binding to monocyte CD44 via production of IL-2 and IFN-gamma

Interactions of the cell surface proteoglycan CD44 with the extracellular matrix glycosaminoglycan hyaluronan (HA) are important during inflammatory immune responses. Our previous studies indicated that monocyte HA binding could be induced by TNF-alpha. Moreover, monocyte HA binding could be markedly up-regulated by culturing PBMC with anti-CD3 (TCR complex) mAbs. The present study was undertaken to identify soluble factors and/or cell surface molecules of activated T lymphocytes that might regulate HA binding to monocytes. Abs to IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-10, IL-15, GM-CSF, IFN-gamma, and TNF-alpha were tested for their effects on anti-CD3 mAb-, Con A-, and PMA/ionomycin-mediated monocyte HA binding in PBMC cultures. Anti-TNF-alpha, anti-IL-2, and anti-IFN-gamma Abs, when added together to PBMC cultures, completely blocked Con A- and partially blocked anti-CD3- and PMA/ionomycin-induced monocyte HA binding. Furthermore, when added together to PBMC cultures, IL-2 and TNF-alpha induced high levels of monocyte HA binding. Likewise, IFN-gamma augmented TNF-alpha-induced monocyte HA binding. To investigate the role of T cell-monocyte direct contact in induction of monocyte HA binding, we studied PMA/ionomycin-activated, paraformaldehyde-fixed CD4(+) T cells in these assays. Fixed, PMA/ionomycin-activated CD4(+) T lymphocytes induced monocyte HA binding, but direct T cell-monocyte contact was not required. Moreover, anti-IFN-gamma and anti-TNF-alpha Abs blocked fixed PMA/ionomycin-activated CD4(+) T cell-induced monocyte HA binding. Taken together, these studies indicate roles for soluble T lymphocyte-derived factor(s), such as IL-2 and IFN-gamma, and a role for monocyte-derived TNF-alpha in Con A-, TCR complex-, and PMA/ionomycin-induced HA binding to monocyte CD44.     

Human thymus-independent antibody response in vitro: III. Precursor frequencies and fine specificity of human B cells stimulated by TNP-Ba in the presence and absence of Con A

Previously, we reported that human B lymphocytes can be stimulated by trinitrophenylated Brucella abortus (TNP-Ba) in vitro to generate T-independent (TI) hapten specific plaque-forming cells (PFC). Furthermore we showed that addition of Con A on Day 3 of culture enhanced the anti-TNP response. In this report the characteristics of the anti-TNP PFC responses elicited by TNP-Ba in the presence or absence of Con A were further defined by estimating precursor frequencies and clone sizes, and by assessing the diversity of the anti-TNP-PFC response using hapten inhibition profiles. Addition of Con A to TNP-Ba-stimulated cultures was associated with a 2.3- to 3.6-fold increase in anti-TNP precursors and was also accompanied by a more vigorous clonal expansion. Fine specificity analysis of anti-TNP PFC revealed that Con A addition resulted in PFC with unique hapten inhibition profiles in that they were less inhibitable by TNP-EACA, TNP-lysine, and DNP-lysine but more inhibitable by DNP-glycine when compared to PFC generated by TNP-Ba alone. These findings suggest that at least some of the additional precursors recruited in the presence of Con A are qualitatively distinct from those activated by TNP-Ba alone since they express different V region gene products.     

Release of lymphokines after infection with Epstein Barr virus in vitro. II. A monocyte-dependent inhibitor of interleukin 1 downregulates the production of interleukin 2 and interferon-gamma in rheumatoid arthritis

Epstein Barr virus (EBV)-infection of normal peripheral blood mononuclear cells (PBMC) in vitro induces IFN-alpha secretion from B cell and natural killer (NK) cell populations, and IFN-gamma secretion from T cells. IFN-gamma depends on prior elaboration of IL 2 and IL 1 that originates from monocytes and NK cells. PBMC from rheumatoid arthritis (RA) patients released moderately elevated levels of IFN-alpha (236 +/- 62 U/ml vs 168 +/- 34 in normals). In contrast, IFN-gamma was significantly lower in RA (88 +/- 34 U/ml vs 209 +/- 32) with an associated deficit in IL 2. A monocyte-dependent factor was shown to be responsible for this deficit, since monocyte depletion of RA cultures normalized the levels of IL 2 and IFN-gamma. Significantly lower levels of IL 1 activity were present in the supernatants of RA PBMC cultures as compared with normal cultures, and this was shown to be associated with presence of a nondialyzable IL 1 inhibitor. This inhibitor was capable of preventing the IL 1-dependent synthesis of IL 2 and IFN-gamma by normal PBMC. Exogenous IL 1 or IL 2 restored the deficient IFN-gamma secretion in RA PBMC. Thus, the deficient ability of RA lymphocytes to control EBV infection may be secondary to impairment of a monocyte-T cell interaction at the level of IL 1.     

Lectin-induced regulation of human thymus-independent anti-TNP responses in vitro

Recently we showed that human hapten-specific T cell-independent (TI) antibody responses could be elicited in vitro with the antigen trinitrophenylated Brucella abortus (TNP-Ba). Although by definition T cells are not required for TI responses, they have also shown capable of regulating such responses in the mouse. In this study we examine the ability of the T cell lectins Con A and PHA to modulate TI responses of human tonsil cells. Addition of the lectins to cultures on day 0 or day 3 resulted in inhibition (greater than 90%) or enhancement (greater than 150%) of the anti-TNP PFC response, respectively. The inhibition was only apparent if the lectins were added at the onset of culture but not 24 hr later, and was T cell-dependent, because T cell-depleted cultures (less than 0.5% E-rosetting cells) could not be inhibited. The enhancement observed was even greater in T cell-depleted cultures and was antigen-specific because TNP-SRBC but not PC-SRBC targets were lysed and the PFC were inhibited by soluble TNP (greater than 70%). This finding suggested that anti-TNP PFC precursors were proliferating more vigorously or that additional, previously antigen-insensitive cells were being recruited in the presence of lectin.     

Excessive production of IFN-gamma in patients with systemic lupus erythematosus and its contribution to induction of B lymphocyte stimulator/B cell-activating factor/TNF ligand superfamily-13B

Expression and immunological significance of IFN-gamma, a pivotal cytokine in murine lupus, have not been clearly demonstrated in human systemic lupus erythematosus (SLE). In the present study we investigated the expression of IFN-gamma in peripheral blood T cells from patients with SLE and its role in the production of the soluble B lymphocyte stimulator (sBLyS). Peripheral blood T cells from patients with SLE expressed significantly larger amounts of IFN-gamma in response to stimulation with anti-CD3 mAb plus anti-CD28 mAb than those from normal controls as shown by three analytical methods, including ELISA, flow cytometry, and quantitative RT-PCR. The ratio of IFN-gamma-producing T cells to effector memory T cells in CD3(+)CD4(+) and CD3(+)CD8(+) populations in patients with SLE was significantly higher than that of normal controls. The T-box expressed in T cells (T-bet) mRNA/GATA-binding protein-3 (GATA-3) mRNA ratio was significantly higher in patients with SLE than in normal controls. T cell culture supernatants from patients with SLE contained significantly higher sBLyS-inducing activity than normal controls; this was almost completely inhibited by the addition of anti-human IFN-gamma mAb. Percentages of BLyS-expressing peripheral blood monocytes in patients with SLE were significantly higher than those of normal controls. Monocytes from patients with SLE produced significantly larger amounts of sBLyS in response to IFN-gamma than those from normal controls. Taken together, these data strongly indicate that the overexpression of IFN-gamma in peripheral blood T cells contributes to the immunopathogenesis of SLE via the induction of sBLyS by monocytes/macrophages, which would promote B cell activation and maturation.     

Evaluation of killing, superoxide anion and nitric oxide production by Leishmania infantum-infected dog monocytes

Protozoa of the genus Leishmania infect reticuloendothelial cells of several mammalian species, including dogs, in which they often give rise to a chronic, not self-healing visceral disease. The parasitocidal mechanism of peripheral blood monocytes towards Leishmania in the dog has not been investigated in detail. Consequently, Leishmania infantum-infected monocyte cultures of healthy dogs were evaluated using the following parameters: (1) phagocytosis and killing capacities; (2) oxidative burst, in terms of superoxide anion (O2-) release, and (3) nitric oxide (NO) activity, in terms of nitrite (NO2-) production in the presence or absence of the NO synthase inhibitor NG-monomethyl-L-arginine (NGMMLA). Parallel experiments were performed on monocytes stimulated with supernatants of concanavalin A-activated PBMC and on unstimulated monocytes. The amount of IFN-gamma in PBMC supernatants used for monocyte activation was determined by a biological assay on a canine Madin Darby cell line. Results demonstrated that phagocytosis, killing capacity and O2- production significantly increased in monocytes stimulated with supernatants, in comparison with unstimulated cells. A positive correlation was observed between the killing capacity, the O2- production and the amount of IFN-gamma in PBMC supernatants employed for monocyte activation. No significant differences were observed in NO production between unstimulated and stimulated cultures, or between the same cultures with and without NGMMLA. Finally, the killing percentage was similar in the presence or absence of NGMMLA, suggesting that in this experimental model peripheral blood dog monocytes lack NO-mediated killing.     

Stimulation of in vitro immunoglobulin production by interferon-alpha

The effect of various natural and recombinant DNA-derived human interferon-alpha (IFN-alpha) on immunoglobulin (Ig) production by human B cells was investigated. The cell populations examined included peripheral blood mononuclear cells (PBMC) and highly purified B cell and helper T cell populations obtained by negative selection by using monoclonal antibodies and a fluorescence-activated cell sorter. In the presence of all forms of IFN-alpha tested, IgG and IgM production by PBMC increased twofold to fourfold. This increase was noted in the absence of pokeweed mitogen (PWM), was not affected by depletion of monocytes, required that IFN-alpha was present early in the culture period, and reached maximal levels around 500 U/ml IFN-alpha. Both IgG and IgM production were affected, but the magnitude of the IgM response was greater. The augmentation of Ig production was noted with the recombinant DNA-derived subtype, IFN-alpha F, two analogs, IFN-alpha Con1 and IFN-alpha Con2, as well as with buffy-coat-derived (leukocyte) IFN-alpha. The recombinant DNA-derived forms of IFN-alpha appeared to differ in their ability to augment Ig production. In the presence of PWM, IFN-alpha Con1 failed to increase Ig production by PBMC. In contrast to these results with PBMC, IFN-alpha Con1 increased the Ig production of purified B cells 10- to 20-fold in the presence of PWM. This increase reached maximal levels around 500 U/ml IFN-alpha Con1. Although purified B cells responded to IFN-alpha and PWM, maximal responses occurred in the presence of low numbers of helper T cells. Cell dilution experiments suggested that the effect observed with purified B cells was the result of the interaction of B cells with residual cells, e.g., helper T cells, remaining in the preparations.     

T cell-monocyte interactions in the production of humoral factors regulating human granulopoiesis in vitro

A variety of monocyte functions are modulated by the T cell lymphokine interferon-gamma (IFN-gamma). We assessed the capacity of IFN-gamma to induce release of granulocyte-monocyte stimulating factors (CSF-GM) from highly purified monocyte preparations. Whereas secretion of CSF-GM by monocytes is negligible in the absence of T cells, CSF-GM secretion is inducible when concentrations of IFN-gamma as low as 10 U/ml are present. This effect could be abrogated by specific neutralizing monoclonal antibody to IFN-gamma and was restricted to monocytes, as resting T cells failed to secrete detectable CSF-GM in response to IFN-gamma. The response of monocyte preparations to IFN-gamma was biphasic in that concentrations greater than 250 U/ml did not induce detectable CSF-GM activity. However, this was shown to be due to the release of a humoral inhibitor of G/M-progenitor cells, and not necessarily due to a lack of CSF-GM secretion.     

IL-12, IFN-gamma, and T cell proliferation to measles in immunized infants

Measles infection in infants is associated with severe complications, and secondary infections are attributed to generalized immunosuppression. Measles binding to its monocyte receptor down-regulates IL-12 which is expected to diminish Th1-like cytokine responses, including IFN-gamma. Whether young infants can be immunized effectively against measles is an important public health issue. We evaluated Ag-specific IL-12, IFN-gamma, and T cell responses of infants at 6 (n = 60), 9 (n = 46), or 12 mo (n = 56) of age and 29 vaccinated adults. IL-12 and IFN-gamma release by PBMC stimulated with measles Ag increased significantly after measles immunization in infants. IL-12 and IFN-gamma concentrations were equivalent in younger and older infants, but IL-12 concentrations were significantly lower in infants than in adults (p = 0.04). IL-12 production by monocytes was down-regulated by measles; the addition of recombinant human IL-12 enhanced IFN-gamma production by PBMC stimulated with measles Ag, but infant T cells released significantly less IFN-gamma than adult T cells under this condition. Of particular interest, the presence of passive Abs to measles had no effect on the specific T cell proliferation or IFN-gamma production after measles stimulation. Cellular immunity to measles infection and vaccination may be limited in infants compared with adults as a result of less effective IFN-gamma and IL-12 production in response to measles Ags. These effects were not exaggerated in younger infants compared with effects in infants who were immunized at 12 mo. In summary, infant T cells were primed with measles Ag despite the presence of passive Abs, but their adaptive immune responses were limited compared with those of adults.     

The anti-T cell monoclonal antibody 9.3 (anti-CD28) provides a helper signal and bypasses the need for accessory cells in T cell activation with immobilized anti-CD3 and mitogens

CD28 is an antigen of 44 kDa which is expressed on the membrane of the majority of human T cells. The present study examines the functional effects of an anti-CD28 monoclonal antibody (mAb 9.3) on T cell activation induced with immobilized anti-CD3 mAb OKT3 or with mitogens, in the absence of accessory cells. To this end, we used blood resting T cells that were completely depleted of accessory cells (monocytes, B cells, and natural killer cells), and consequently did not respond to recombinant interleukin-2 (rIL-2), to immobilized OKT3, to PHA, or to Con A. Addition of mAb 9.3 to the cultures enhanced IL-2 receptor expression (Tac antigen) on PHA- or immobilized OKT3-stimulated T cells and induced IL-2 receptors on Con A-stimulated T cells. Moreover, addition of mAb 9.3 to cultures of T cells stimulated with PHA, Con A, or immobilized OKT3 resulted in IL-2 production. Soluble mAb 9.3 was a sufficient helper signal for T cell proliferation in response to PHA or immobilized OKT3. Crosslinking of mAb 9.3 by culture on anti-mouse IgG-coated plates enhanced the helper effect and was an essential requirement for the induction of T cell proliferation in response to Con A. No other anti-T cell mAb (anti-CD2, -CD4, -CD5, -CD7, -CD8) was found to provide a complete accessory signal for PHA or Con A stimulation of purified T cells. T cell proliferation induced by the combination of PHA and mAb 9.3 was strongly inhibited by the anti-IL-2 receptor mAb anti-Tac. In conclusion, mAb 9.3 can provide a signal bypassing monocyte requirement in T cell activation with immobilized OKT3, PHA, and Con A, resulting in an autocrine IL-2-dependent pathway of proliferation.     

Bacterial lipopolysaccharide-induced interferon-gamma production: roles of interleukin 1 and interleukin 2

Bacterial lipopolysaccharide (LPS) induced human peripheral blood mononuclear cells (PBMC) to produce interferon-gamma (IFN-gamma). Monocytes play a mandatory accessory role in this process, because purified T lymphocytes failed to produce IFN-gamma in response to LPS and the addition of 2% monocytes to T cell cultures resulted in an optimal LPS-induced IFN-gamma production. IFN-gamma production was abolished in the presence of monoclonal antibodies specific for HLA-DR antigen. Addition of exogenous interleukin 2 (IL 2) markedly enhanced IFN-gamma secretion by PBMC induced with LPS. The addition of anti-Tac antibody specific for IL 2 receptors abrogated IFN-gamma production, suggesting that an interaction of IL 2 with IL 2 receptors was involved. By using a specific antibody binding assay, LPS was shown to amplify IL 2 receptor expression on PBMC, whereas exogenous IL 2 showed only a negligible enhancing effect on the expression of its own receptors. Interleukin 1 (IL 1), a product of LPS-stimulated monocytes, potentiated IL 2-induced IFN-gamma production in the absence of LPS. Neither IL 1 nor IL 2 alone induced IFN-gamma production in purified T lymphocyte cultures. When added together, however, substantial levels of IFN-gamma were induced. An enhanced IL 2 receptor expression on T cells was also demonstrated as a result of the combined action of IL 1 and IL 2. These results suggest that induction of IFN-gamma by LPS is due mainly to the generation of IL 1 and an enhanced expression of IL 2 receptors.     

Virus-induced interferon alpha/beta (IFN-alpha/beta) production by T cells and by Th1 and Th2 helper T cell clones: a study of the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta on functions of different T cell populations

Spleen cells, resting T cells, activated T cells, and T cell clones characterized as type 1 (Th1) and type 2 (Th2) were investigated for their ability to produce interferon (IFN) following in vitro culture with Newcastle disease virus (NDV). All of the above cell populations, including both Th1 and Th2 T cell clones, produced high levels of IFN following in vitro culture with NDV. This IFN was characterized as a mixture of IFN-alpha and IFN-beta with IFN-alpha being the predominate species of IFN contained in the mixture. IL-2 greatly enhanced the production of IFN-alpha/beta by all cell populations in response to NDV. These different T cell populations responded very differently to the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta. IFN-alpha/beta was shown to be a potent inhibitor of Con A or IL-2-induced proliferation of different T cell populations. This inhibition was not associated with a reduction in lymphokine production since spleen cells or Th1 T cell clones cultured with Con A and IFN-alpha/beta had no decrease in IL-2 or IFN-gamma production when compared to Con A-stimulated control cultures. IFN-gamma had little to no inhibitory activity on Con A-induced proliferation of spleen cells. In fact, Con A-induced proliferation was usually enhanced by IFN-gamma when nylon wool-enriched T cells were assessed. Different results were observed when IFN-gamma and IFN-alpha/beta were investigated for their ability to inhibit IL-2-induced proliferation of different T helper cell clones. IFN-gamma and IFN-alpha/beta were both capable of inhibiting IL-2-induced proliferation of T cell clones characterized as type 2 (Th2). In contrast, IFN-gamma had no effect on IL-2-induced proliferation of Th1 clones. IFN-alpha/beta, however, inhibited IL-2-induced proliferative responses of both Th1 and Th2 T cell clones. These results document the facts that (1) IFN-gamma and IFN-alpha/beta differ in their immunoregulatory actions, (2) different T cell subpopulations vary in their susceptibility to IFN-gamma regulation, and (3) virus induction of IFN-alpha/beta appears to be a ubiquitous function associated with different T cell populations.     

Influence of recombinant IL-4, IFN-alpha, and IFN-gamma on the production of human IgE-binding factor (soluble CD23)

This study documents the influence of rIL-4, IFN-gamma, and IFN-alpha on the production of IgE-BF and the expression of lymphocyte receptor for IgE or CD23 Ag (Fc epsilon R II) by human mononuclear cells. IL-4 increases the secretion of IgE-binding factor (BF) by highly purified B lymphocytes, adherent cells, and U937 monoblastic cells. The effect of IL-4 on purified B cells is augmented by costimulating the cells with F(ab')2 anti-IgM. IFN-gamma, IL-2, IL-1-alpha, or IL-1 beta and the low m.w. B cell growth factor have no effect on IgE-BF production by purified B cells even when they are used in combination with anti-IgM. Stimulation of purified T cells with IL-4 or IL-4 plus PMA leads to the production of very small amounts of IgE-BF that might well be derived from the contaminating non-T cells. IFN-gamma increases IgE-BF synthesis by unfractionated PBMC, T cell-depleted PBMC, adherent cells, and U937 cells suggesting that it induces monocytes to release IgE-BF, IFN-gamma suppresses the IL-4-induced Fc epsilon R II expression and IgE-BF production by highly purified B cells but not by PBMC or their T cell-depleted fractions. IFN-alpha inhibits IgE-BF production by IFN-gamma-stimulated PBMC and by IL-4-stimulated cells suggesting that it exerts its effect on B cells and on monocytes. Moreover IFN-alpha suppresses the IL-4-induced expression of Fc epsilon R II on B cells. Both IFN-alpha and IFN-gamma suppress the synthesis of IgE by PBMC in response to IL-4. Taken collectively the results indicate that: 1) IL-4 induces IgE-BF production by both B cells and monocytes, 2) IFN-gamma stimulates IgE-BF synthesis by monocytes but suppresses its production by IL-4-stimulated B cells, and finally 3) IFN-alpha inhibits IgE-BF synthesis in response to either IFN-gamma or IL-4.     

Accessory cell requirements for the mixed-leukocyte reaction and polyclonal mitogens, as studied with a new technique for enriching blood dendritic cells

Human blood dendritic cells can be enriched to 40-80% purity by a new technique that is simpler, provides greater yields than prior methods, and resolves other populations that are enriched in monocytes and B and T lymphocytes. The procedure involves separation over two Percoll gradients after 0 and 2 days of culture, followed by removal of contaminating monocytes by panning on plates coated with human Ig. The resultant dendritic cell-enriched fraction is 10 times or more potent than the monocyte-enriched populations in stimulating T-cell proliferative responses to alloantigens and to Con A. Small B lymphocytes are inactive in both systems. Dendritic cells do not initiate mitogenesis to anti-CD3 monoclonal antibodies, a response for which the monocyte appears to be the critical accessory cell.     

Cytokine regulation of localized inflammation. Induction of activated B cells and IL-6-mediated polyclonal IgG and IgA synthesis in inflamed human gingiva

It is well established that increased numbers of plasma cells occur in the localized tissues of chronic inflammatory diseases such as adult periodontitis, and enzymatic isolation has shown that most B lineage cells produce IgG-subclass with some IgA-subclass responses. It would be of importance to determine if excess production of cytokines in the localized lesion account for these responses and in the present study we have assessed gingival mononuclear cell (GMC) supernatants for cytokines that activate B cells including IL-6R expression and for levels of IL-6 present. Inasmuch as limited numbers (approximately 1 to 3 x 10(6) cells) of GMC were obtained from surgically removed tissues (approximately 400 mg), we have focused on the analysis of IL-6 production by GMC in this study. Further, initial evidence of additional cytokines that are produced by GMC and induce expression of IL-6R on resting B cells has been obtained. The GMC and PBMC from individual patients were cultured in the presence (or absence) of Con A. Higher levels of IL-6 were produced spontaneously by GMC when compared with Con A-stimulated PBMC. When PBMC cultures were supplemented with GMC supernatants obtained from the same patient, high numbers of spot-forming cells (SFC), mainly of IgG followed by IgA isotype, were seen. The induction of SFC by GMC supernatants was inhibited by incubation with a goat anti-human IL-6 antibody. When the effect of GMC supernatants on subclasses of PBMC SFC was determined, the response was IgG1 greater than IgG2 greater than IgG3 = IgG4 and IgA1 greater than IgA2, a pattern remarkably similar to the distribution of plasma cells in the GMC itself. To assess for cytokines in GMC supernatants that mediated B cell activation, supernatants containing anti-IL-6 were cultured with PBMC or purified B cells for 72 h. This treatment induced small proliferative B cell responses and elevated expression of IL-6R on B cells, but did not induce SFC responses. Further, incubation of B cells with GMC supernatants induced resting B cells (G0/G1) to enter the cell cycle (S and G2/M). Addition of human rIL-6 to these cultures on day 3 restored IgG- and IgA-subclass SFC responses by day 7. Cytokine-induced IL-6R expression also occurred in vivo because freshly isolated GMC expressed high levels of this receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

MIF-like activity of natural and recombinant human interferon-gamma and their neutralization by monoclonal antibody

By utilizing elutriation-purified human monocytes, we found that human interferon (IFN) inhibits monocyte migration in a manner similar to migration inhibitory factor (MIF) and does it without demonstrable cytotoxicity. We observed that human IFN-gamma is 10 to 300 times more potent in its MIF activity than is IFN-alpha and that monoclonal antibodies (MoAb) can be used to distinguish between them. Studies with recombinant IFN-gamma indicate that the migration inhibition seen with natural IFN-gamma is due to IFN-gamma itself and is not due to co-purification of another lymphokine with the natural IFN-gamma. Although interferons exhibit MIF activities, there are apparently other cytokines, without antiviral activity, that also have MIF activities. MIF from the lymphoblastoid cell line RPMI 1788 was not neutralized by MoAb to IFN. However, MIF activity in supernatant fluid from human peripheral blood lymphocyte cultures stimulated with Con A-Sepharose was completely neutralized with MoAb anti-IFN-gamma. These data indicate that MIF is really a family of cytokines that inhibit macrophage/monocyte migration and that the major portion of MIF activity associated with crude supernatant of mitogen-stimulated lymphocytes is due to IFN-gamma.     

Antibodies against the CD44 p80, lymphocyte homing receptor molecule augment human peripheral blood T cell activation

The CD44 inhibitor Lutheran [In(Lu)]-related p80 molecule has recently been shown to be identical to the Hermes-1 lymphocyte homing receptor and to the human Pgp-1 molecule. We have determined the effect of addition of CD44 antibodies to in vitro activation assays of PBMC. CD44 antibodies did not induce PBMC proliferation alone, but markedly enhanced PBMC proliferation induced by a mitogenic CD2 antibody pair or by CD3 antibody. CD44 antibody addition had no effect upon PBMC activation induced by PHA or tetanus toxoid. CD44 antibody enhancement of CD2 antibody-induced T cell activation was specific for mature T cells as thymocytes could not be activated in the presence of combinations of CD2 and CD44 antibodies. CD44 antibody enhancement of CD2-mediated T cell triggering occurred if CD44 antibody was placed either on monocytes or on T cells. In experiments with purified monocyte and T cell suspensions, CD44 antibodies A3D8 and A1G3 augmented CD2-mediated T cell activation by three mechanisms. First, CD44 antibody binding to monocytes induced monocyte IL-1 release, second, CD44 antibodies enhanced the adhesion of T cells and monocytes in CD2 antibody-stimulated cultures, and third, CD44 antibodies augmented T cell IL-2 production in response to CD2 antibodies. Thus, ligand binding to CD44 molecules on T cells and monocytes may regulate numerous events on both cell types that are important for T cell activation. Given that recent data suggest that the CD44 molecule may bind to specific ligands on endothelial cells (vascular addressin) and within the extracellular matrix (collagen, fibronectin), these data raise the possibility that binding of T cells to endothelial cells or extracellular matrix proteins may induce or up-regulate T cell activation in inflammatory sites.     

Evaluation of the in vitro production of interferon gamma and other lymphokines in uremic patients

It has been suggested that a deficient immune response can be responsible at least partially for the high risk of infections and neoplasia in uremic patients. Since interferon (IFN) is critical to the immune response, we have evaluated the in vitro production of IFN-gamma and other lymphokines by peripheral blood mononuclear cells (PBMC) drawn from patients with end-stage renal disease and appropriate controls. We have correlated production of lymphokines by these cells with proliferative response to different mitogens. It was found that the secretion of IFN-gamma in response to all three mitogens was elevated in these patients compared with the control group. This elevation was significant with both phytohemagglutin and staphylococcal enterotoxin A, but not with Con A. No significant difference was observed in production of lymphotoxins, IL-2, and leukocyte migration inhibition responses. In contrast the proliferative response appeared diminished in the PBMC of uremic patients. We concluded that defective lymphokine generation is not a major immunological problem in patients with end-stage renal disease. Indeed, they appear to release excess amount of IFN-gamma which is known to be a macrophage-activating factor. It is suggested that high IFN-gamma activity could enhance the secretion of IL-1 or endogenous pyrogen and result in development of febrile reactions in dialysis patients.     

Human double-negative T cells in systemic lupus erythematosus provide help for IgG and are restricted by CD1c

To understand the mechanism of T cell help for IgG production in systemic lupus erythematosus (SLE) we investigated the response of CD4- and CD8-negative (double-negative (DN)) T cells because 1) DN T cells are present at unusually high frequency in patients with SLE and can induce pathogenic autoantibodies; 2) the DN T cell repertoire includes cells restricted by CD1 Ag-presenting molecules; and 3) CD1c is expressed on a population of circulating B cells. We derived DN T cell lines from SLE patients and healthy individuals. In the presence of CD1(+) APCs, DN T cell lines from SLE patients produced both IL-4 and IFN-gamma, whereas DN T cells from healthy donors produced IFN-gamma, but no IL-4. In general, cells from patients with highly active disease produced high levels of IFN-gamma; cells from those with little activity produced high IL-4. Coculture of CD1c-directly reactive T cells from healthy donors with CD1c(+) B cells elicited IgM Abs, but little or no IgG. In contrast, CD1c-directly reactive T cells from SLE patients induced isotype switching, with a striking increase in IgG production. Neutralizing Abs to CD1c inhibited the ability of DN T cells to induce IgG production from CD1c(+) B cells, further indicating that CD1c mediated the T and B cell interaction. IgG production was also inhibited by neutralizing Abs to IL-4, correlating with the cytokine pattern of DN T cells derived from these patients. The data suggest that CD1c-restricted T cells from SLE patients can provide help to CD1c(+) B cells for IgG production and could therefore promote pathogenic autoantibody responses in SLE.     

Augmentation of phorbol ester-induced T cell proliferation by agents which raise intracellular cyclic adenosine monophosphate.

Although raising intracellular cyclic adenosine monophosphate (cAMP) levels is generally considered to be inhibitory on the mitogen-induced T cell proliferation, in this study we have shown that the addition of either dbcAMP (50 microM) or cholera toxin (1 ng/ml) resulted in an increase in [3H]thymidine uptake in PBMC cultures stimulated with phorbol ester, 12-tetradecanoylphorbol 13-acetate (TPA), or with a combination of TPA plus anti-CD3 mAb (mAb 235). In contrast, under similar culture conditions, the phytohemagglutinin-P (PHA-P) response was inhibited by these agents as has been reported. The augmentative effect of dbcAMP in PBMC cultures was due to an increase in IL-2 production and not to increased in IL-2R-alpha chain expression. The enhancing effect of dbcAMP and CT observed with PBMC was monocyte dependent and not seen with purified T cell preparations. The addition of monocytes reconstituted the ability of intracellular cAMP elevating agents to augment the T cell response to TPA with and without mAb to CD3. The monocytes mediate their action via soluble factor(s) with molecular weight (m.w.) of more than 10 kDa. Neither rIL-1, rIL-6, nor rTNF-alpha have any augmentative effect as contrast with the supernatant from treated monocytes. Taken together, our results indicate that cAMP can play a positive regulatory role in T cell proliferation due to factor(s) secreted by dbcAMP-treated monocytes resulting in increased IL-2 synthesis in T cells.