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.

Genetic deletion of the tumor necrosis factor receptor p60 or p80 abrogates ligand-mediated activation of nuclear factor-kappa B and of mitogen-activated protein kinases in macrophages.

Tumor necrosis factor (TNF) is a pleiotropic cytokine known to regulate cell growth, viral replication, inflammation, immune system functioning, angiogenesis, and tumorigenesis. These effects are mediated through two different receptors, TNFR1 and TNFR2 (also called p60 and p80, respectively), with p60 receptor being expressed on all cell types and p80 receptor only on cells of the immune system and on endothelial cells. Although the role of p60 receptor in TNF signaling is well established, the role of p80 is less clear. In this report, by using macrophages derived from wild-type mice (having both receptors) and mice in which the gene for either p60 (p60(-/-)), or p80 (p80(-/-)), or both (p60(-/-) p80(-/-)) receptor have been deleted, we have redefined the role of these receptors in TNF-induced activation of nuclear factor (NF)-kappa B and of mitogen-activated protein kinases. TNF activated NF-kappa B in a dose- and time-dependent manner in wild-type macrophages but not in p60(-/-), p80(-/-), or p60(-/-) p80(-/-) macrophages. These results correlated with the I kappa B alpha degradation needed for NF-kappa B activation. We also found that TNF activated c-Jun N-terminal protein kinase in a dose- and time-dependent manner in wild-type macrophages but not in p60(-/-), p80(-/-), or p60(-/-) p80(-/-) macrophages. TNF activated p38 MAPK and p44/p42 MAPK in wild-type but not in p60(-/-), p80(-/-), or p60(-/-) p80(-/-) macrophages. TNF induced the proliferation of wild-type macrophages, but for p60(-/-) and p80(-/-) macrophages proliferation was lower, and in p60(-/-) p80(-/-) it was absent. Overall, our studies suggest that both types of TNF receptors are needed in macrophages for optimum TNF cell signaling.

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.

CD40 signaling of monocyte inflammatory cytokine synthesis through an ERK1/2-dependent pathway. A target of interleukin (il)-4 and il-10 anti-inflammatory action.

Ligation of CD40 on monocytes through its interaction with CD40 ligand (CD154) present on activated T helper cells, results in activation of monocyte inflammatory cytokine synthesis and rescue of monocytes from apoptosis induced through serum deprivation. Both of these consequences of CD40 stimulation have been shown to be dependent on the induction of protein tyrosine kinase activity. CD40-mediated activation of protein tyrosine kinase activity and subsequent inflammatory cytokine production are abrogated by treatment of monocytes with the T helper type 2 cytokines interleukin 4 (IL-4) and interleukin 10 (IL-10). In the current study we demonstrate that stimulation of monocytes through CD40 resulted in the phosphorylation and activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinases, whereas phosphorylation of mitogen-activated protein kinases family members p38 and c-Jun N-terminal kinase was not observed in response to this stimuli over the time course examined. PD98059, an inhibitor of the upstream activator of ERK1/2, the MAP/ERK kinase MEK1/2, suppressed IL-1beta and tumor necrosis factor-alpha production in a dose-dependent fashion. Pretreatment of monocytes with IL-4 and IL-10 inhibited CD40-mediated activation of ERK1/2 kinase activity when used individually, and are enhanced in effectiveness when used in combination. Together, the data demonstrate that CD40-mediated induction of IL-1beta and tumor necrosis factor-alpha synthesis is dependent on a MEK/ERK pathway which is obstructed by signals generated through the action of IL-4 and IL-10.

Membrane potassium channels and human bladder tumor cells: II. Growth properties.

These experiments were done to determine the effect of glibenclamide and diazoxide on the growth of human bladder carcinoma (HTB-9) cells in vitro. Cell growth was assayed by cell counts, protein accumulation, and 3H-thymidine uptake. Glibenclamide added at 75 and 150 microM for 48 hr reduced cell proliferation. Dose-inhibition curves showed that glibenclamide added for 48 hr reduced cell growth at concentrations as low as 1 microM (IC50 = 73 microM) when growth was assayed in the absence of added serum. This microM-effect on cell growth was in agreement with the dose range in which glibenclamide decreased open probability of membrane KATP channels. Addition of glibenclamide for 48 hr also altered the distribution of cells within stages of the cell cycle as determined by flow cytometry using 10(-5) M bromodeoxyuridine. Glibenclamide (100 microM) increased the percentage of cells in G0/G1 from 33.6% (vehicle control) to 38.3% (P < 0.05), and it reduced the percentage of cells in S phase from 38.3% to 30.6%. On the other hand, diazoxide, which opens membrane KATP channels in HTB-9 cells, stimulated growth measured by protein accumulation, but it did not increase the cell number. We conclude that the sulfonylurea receptor and the corresponding membrane KATP channel are involved in mechanisms controlling HTB-9 cell growth. However, KATP is not rate-limiting among the signaling mechanisms or molecular switches that regulate the cell cycle.

IL-4 and IL-10 modulation of CD40-mediated signaling of monocyte IL-1beta synthesis and rescue from apoptosis.

Previous studies have demonstrated that the interaction of CD40 on monocytes with CD40 ligand, present on activated CD4+ T cells, induces monocyte inflammatory cytokine synthesis and rescues monocytes from apoptosis. These findings suggest a role for CD40 signaling of monocyte activation in the maintenance and/or exacerbation of nonseptic (e.g., autoimmune) inflammatory responses. In the present study the effects of the modulatory cytokines IL-4 and IL-10 on CD40-mediated signaling of monocyte IL-1beta synthesis and rescue from apoptosis were examined. Both IL-4 and IL-10 decreased CD40-dependent IL-1beta synthesis in a dose-dependent manner individually and synergized in this effect when used concurrently, with minimal effect on CD40 surface expression. CD40 signaling of IL-1beta synthesis was shown to be dependent on the induction of protein tyrosine kinase (PTK) activity, and both IL-4 and IL-10 diminished CD40-mediated tyrosine phosphorylation of monocyte cellular proteins. However, IL-4, but not IL-10, blocked CD40-mediated rescue from apoptosis, an event that we have demonstrated previously to be dependent on PTK activity as well. Together these results suggest that in monocytes 1) both IL-4 and IL-10 target CD40-induced PTK activity in the down-regulation of IL-1beta synthesis; and 2) IL-4 and IL-10 have divergent effects on the CD40 signaling pathway, in that these cytokines are synergistic with respect to their abilities to inhibit CD40-mediated IL-1beta synthesis and differ in their abilities to block CD40-mediated rescue from apoptosis.

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.

Multifunctional cytokine expression by human mast cells: regulation by T cell membrane contact and glucocorticoids.

Human mast cells readily release a variety of mediators, including cytokines, in response to IgE receptor crosslinking, but the mechanisms governing the expression of cytokines are still unclear. Using a human mast cell line, HMC-1, we show expression of cytokine transcripts as early as 2 h after activation with ionomycin and phorbol myristate acetate (PMA). Resting HMC-1 cells expressed transcripts for interleukin-1 receptor antagonist (IL-1RA), IL-2, IL-4, IL-5, GM-CSF, and weakly for IL-8, and stimulation with ionomycin and PMA induced additional transcripts for IL-6 and IL-13 and upregulated expression of IL-8 transcripts. HMC-1 cells secreted IL-4, IL-8, and GM-CSF protein after activation and dexamethasone significantly inhibited the production of these cytokines. Of significance is the finding that the addition of membranes purified from activated T cells to mast cell cultures induced transcripts selectively for IL-8 and none for other proinflammatory cytokines. Flow cytometry revealed that resting HMC-1 cells express CD40, a molecule involved in contact-dependent signaling of monocytes and B cells by T cells. However, activation of HMC-1 by anti-CD40 antibody did not induce IL-8 gene expression or protein production. This study demonstrates that human mast cells are capable of expressing multiple cytokines that can be inhibited by glucocorticoids. It also raises the possibility that T cells may activate mast cell cytokine synthesis by novel contact-dependent mechanisms. This phenomenon of T cell regulation of mast cell function requires further study.

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 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.

Impaired T cell-mediated macrophage activation in CD40 ligand-deficient mice.

The expression of the ligand for CD40 (CD40L) is critical for induction of T cell-dependent Ab responses. To examine how critical the expression of CD40L is for induction of cell-mediated immune responses, the ability of T cells from CD40L knockout mice to activate macrophage effector function was assessed. CD4+ T cells from CD40L-knockout mice were fourfold less effective than +/+ T cells in activating the nitric oxide response in allogeneic macrophages. CD40L-knockout T cells that were fixed with paraformaldehyde after a 6-h activation period, a time point at which CD40L dominates the macrophage-activating capability of the T cell, could activate neither macrophage production of inflammatory cytokines (TNF-alpha) nor generation of reactive nitrogen intermediates. After 24 h of activation, however, both CD40L-knockout and +/+ T cells could induce similar but weak responses from the macrophages. This study demonstrates that animals deficient in CD40L expression display a deficiency in T cell-dependent macrophage-mediated immune responses.

Indomethacin does not alter natural killer cell response to 2.5 h of running.

The effect of 2.5 h of treadmill running at 75.6 +/- 0.9% maximal O2 uptake (VO2max) on natural killer (NK) cell cytotoxic activity (NKCA) was investigated in 22 experienced marathon runners (VO2max 57.9 +/- 1.1 ml.kg-1.min-1, age 38.7 +/- 1.5 yr). Blood samples were taken before (0715) and immediately after exercise (1000), with three more samples taken during 6 h of recovery (1130, 1300, and 1600). Ten sedentary controls (VO2max 34.7 +/- 1.0 ml.kg-1.min-1, age 45.3 +/- 2.3 yr) sat in the laboratory during testing and had their blood sampled at the same time points. The pattern of change in NKCA over time was significantly different between groups [F(4,27) = 6.53; P = 0.001], with the runner's NKCA dropping 51-61% below preexercise levels throughout 6 h of recovery. Preincubation of blood mononuclear cells in vitro with indomethacin had no effect on the difference in pattern of change in NKCA between groups [F(4,17) = 8.59; P = 0.001] and did not attenuate the postexercise reduction in the runners. When NKCA was adjusted on a per-NK cell basis, group differences and the postexercise decline in NKCA were eliminated [F(4,80) = 0.65; P = 0.63]. Serum cortisol and plasma epinephrine in the runners were elevated relative to control subjects during recovery from exercise, but no significant correlation with changes in NK cells or NKCA was found. These data indicate that NKCA is decreased significantly during recovery from 2.5 h of running due to a numerical redistribution of NK cells.

Lymphocyte proliferative response to 2.5 hours of running.

The effect of 2.5 h of treadmill running at 75.6 +/- 0.9% VO2max on circulating leukocyte and lymphocyte subpopulations, epinephrine and cortisol concentrations, and the Con A-induced lymphocyte proliferative response was investigated in 22 experienced marathon runners (VO2max 57.9 +/- 1.1 ml.kg-1.min-1, age 38.7 +/- 1.5 yrs). Blood samples were taken 15 min before (07.15h) and immediately after exercise (10.00h), with three more samples taken during 6h of recovery (11.30, 13.00, 16.00h). Ten sedentary controls (34.7 +/- 1.0 ml.kg-1.min-1, 45.3 +/- 2.3 yrs) sat in the laboratory during testing and had their blood sampled at the same time points. Serum cortisol was elevated relative to controls for more than 3 h post-exercise, and correlated significantly with the 3-h post-exercise, and correlated neutrophil/lymphocyte ratio (r = 0.68, p < 0.001). The concanavalin A- (Con A) induced lymphocyte proliferative response was decreased relative to controls for more than 3 h post-exercise, and except for the immediate post-exercise time point, tended to parallel the decrease in T cell (CD3+) concentrations.

Immune function in marathon runners versus sedentary controls.

Marathon runners (N = 22) who had completed at least seven marathons (X +/- SEM = 23.6 +/- 5.7) and had been training for marathon race events for at least 4 yr (12.3 +/- 1.3) were compared with sedentary controls (N = 18). Although the two groups were of similar age (38.7 +/- 1.5 and 43.9 +/- 2.2 yr, respectively) and height, the marathon runners were significantly leaner and possessed a VO2max 60% higher than that of the controls. Neutrophil counts tended to be lower in the group of marathoners, while other leukocyte and lymphocyte subsets were similar to controls. Mitogen-induced lymphocyte proliferation did not differ between groups. Natural killer cell cytotoxic activity (NKCA) was significantly higher in the marathoners versus controls (373 +/- 38 vs 237 +/- 41 total lytic units, respectively, a 57% difference, P = 0.02). For all subjects combined (N = 40) and within the group of marathon runners (N = 22), percent body fat was negatively correlated with NKCA (r = -0.48, P = 0.002; r = -0.49, P = 0.019, respectively), and age was negatively correlated with Con A-induced lymphocyte proliferation (r = -0.41, P = 0.009; r = -0.53, P = 0.011, respectively). These data indicate that NKCA but not mitogen-induced lymphocyte proliferation is higher in marathon runners relative to sedentary controls.

The acute immune response to exhaustive resistance exercise.

Ten young male adults (mean age 46.9 +/- 1.2 yrs) with 9.2 +/- 1.4 years of weight training experience and the ability to parallel squat at least 1.5 times their body mass were selected as subjects. The exercise session consisted of sets of 10 repetitions at 65% 1-RM of the parallel leg squat, with a cadence of one rep every 6 sec and 3 min rest between sets, to muscular failure. The average subject lifted a total of 9711 +/- 1576 kg during 98 +/- 14 reps for a total work output of 72.5 +/- 10.5 kJ before muscular failure occurred. Mean oxygen consumption during exercise was 1.58 +/- 0.06 l/min at 42.5 +/- 2.0% peak VO2. A strong leukocytosis, lymphocytosis, and lymphocytopenia, similar to what has been reported following high-intensity cardiorespiratory exercise, were measured following leg squat exercise. Con A-stimulated lymphocyte proliferation (unadjusted) rose 50% above preexercise levels (p = 0.07), but when these data were adjusted on a per T cell (CD3+) basis, no change from rest was observed. Natural killer cell cytotoxic activity (NKCA), when adjusted on a per NK cell (CD56+) basis, was decreased about 40% below preexercise levels for at least 2 h post-exercise. No significant increase in cortisol was seen after exercise, although norepinephrine and epinephrine increased moderately (465% and 133%, respectively), immediately following exercise. The data demonstrate that leg squat exercise to muscular failure results in a very similar immune response to that associated with intense endurance exercise, despite a lower mean oxygen consumption and only a moderate hormonal response.

Immune function in athletes versus nonathletes.

The purpose of this study was to compare natural killer cell cytotoxic activity (NKCA) and Con A-induced lymphocyte proliferation (T cell function) in athletes versus nonathletes, with measurement of natural killer (NK) and T cells to allow a comparison on a "per-cell" adjusted basis. Eighteen young male endurance athletes (10 runners and 8 cyclists) with a mean VO2max of 70.7 +/- 1.3 ml.kg-1.min-1 and 6.6 +/- 0.8 years of competitive experience were compared with 11 nonathletic male adults (47.6 +/- 3.1 ml.kg-1.min-1). Concentrations of circulating leukocyte and lymphocyte subsets, including NK and T cells, were not significantly different between groups. NKCA and T cell function also did not differ between groups, whether expressed unadjusted or adjusted on a per-cell basis. For all subjects combined, both NKCA and T cell function were unrelated to VO2max (r = 0.005, p = 0.98; r = 0.007, p = 0.97, respectively). These data do not support the contention that immune function, as measured in this study, is altered in endurance athletes.

Moderate exercise training and natural killer cell cytotoxic activity in breast cancer patients.

Sixteen female breast cancer patients who had been diagnosed (3.0 +/- 1.2 years previous to the study) and undergone surgery, chemotherapy, and/or radiation treatment were randomly assigned to exercise and nonexercise groups. Pre- and post-study measurements were taken for aerobic performance, leg strength, and concentrations of circulating lymphocyte subsets and natural killer cell cytotoxic activity (NKCA). Exercise training consisted of 60 minutes of supervised weight training and aerobic activity three times each week for eight weeks. Although subjects in the exercise groups demonstrated some modest improvement in the various aerobic and strength tests, NKCA and concentrations of circulating T and NK cells were not significantly altered relative to the nonexercise group. This study suggests that moderate exercise over an eight-week period has no significant effect on the function of in vitro natural killer cells in breast cancer patients.

T cell--vascular smooth muscle cell interactions: antigen-specific activation and cell cycle blockade of T helper clones by cloned vascular smooth muscle cells.

Histological observations have demonstrated the presence of T lymphocytes in atherosclerotic plaques, often in close association with vascular smooth muscle cells (VSMC). We have examined the interaction occurring between cloned murine VSMC and histocompatibility-matched, antigen-specific Th1 and Th2 cell lines. Incubation of either Th1 or Th2 cells with antigen-pulsed VSMC resulted in the formation of T cell-VSMC conjugates accompanied by morphological changes in both cell types. This interaction resulted in an antigen-dependent activation of IL-2 receptor expression by the Th cells, demonstrating the ability of cloned VSMC to process and present antigen through the exogenous pathway. However, although the T cells were activated to express IL-2 receptors by antigen-pulsed VSMC, they were unable to progress through cell cycle. The secretion of an inhibitory mediator by VSMC was suggested by the observations that (1) fixation of the VSMC's eliminated the inhibitory signal and (2) the supernatants of IFN gamma-primed VSMC displayed similar inhibitory activity. The inhibitory effect could not be abrogated with indomethacin or an inhibitor of the generation of reactive nitrogen intermediates, indicating that prostaglandin synthesis and/or nitric oxide production are not solely responsible for the inhibition of proliferation. Flow cytometric cell cycle analysis revealed that VSMC delivered signals resulting in a late G1 blockade of T cell cycle progression. Mitogen responses of purified primary T cells are also dramatically inhibited by IFN gamma-treated VSMC, despite significant IL-2 production. Our data depict a complex and intimate T cell-VSMC interaction and suggest that mutual activation events may occur.

Role of the CD40-CD40 ligand interaction in CD4+ T cell contact-dependent activation of monocyte interleukin-1 synthesis.

Most studies of the induction of cytokine synthesis in monocytes have employed an exogenous triggering agent such as lipopolysaccharide. However, in nonseptic inflammatory responses (e.g. rheumatoid arthritis) monocyte activation occurs as a result of T cell-generated signals. In previous reports, we and others have demonstrated that contact-dependent T cell-generated signals are capable of contributing to macrophage activation. We have shown that plasma membranes from anti-CD3 activated purified peripheral CD4+ T cells (TmA) but not from resting CD4+ cells (TmR) induce monocytes to synthesize interleukin (IL)-1 in the absence of co-stimulatory cytokines. Studies to determine the expression kinetics of the molecule(s) unique to activated CD4+ T cells which interact with monocytes to induce IL-1 revealed that optimal expression occurred at 6 h post activation. This matched the previously reported kinetics of expression of CD40 ligand (CD40L) on activated peripheral T cells, implicating the CD40-CD40L interaction as a candidate for the initiator of the IL-1 signaling event. The ability of TmA to induce IL-1 synthesis in resting monocytes could be markedly reduced by addition of a monoclonal anti-CD40L antibody, 5c8. In addition, a monoclonal anti-CD40 IgM (BL-C4) proved dramatic in its ability to induce resting monocytes to synthesize IL-1. In summary, these results demonstrate that the CD40-CD40L interaction provides a critical component of CD4+ T cell contact-dependent activation of monocyte IL-1 synthesis.