Outer membrane vesicles from Neisseria meningitidis: effects on cytokine production in human whole blood

The Norwegian group B meningococcal outer membrane vesicle (OMV) vaccine consists of outer membrane proteins (OMPs) as main antigens with significant amounts of lipopolysaccharide (LPS; 5-9% relative to protein). We have studied the ability of this OMV vaccine preparation to induce secretion of pro-inflammatory cytokines, tumour necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), interleukin 6 (IL-6), interleukin 8 (IL-8) and anti-inflammatory cytokines, interleukin 4 (IL-4), interleukin 10 (IL-10) and interleukin 13 (IL-13) in a human whole blood model. Plasma levels of TNF-alpha, IL-1beta, IL-6 and IL-8 were massively increased; mean peak levels of TNF-alpha 44 696+/-7764, IL-1beta 38 043+/-5411, IL-6 10 057+/-1619 and IL-8 30 449+/-5397 pg/ml were obtained with an OMV-LPS concentration of 1 microg/ml; corresponding levels in control plasmas were below the detection limit of the assay. Mean maximal level of IL-10 (2540+/-144 pg/ml) was obtained at OMV-LPS concentration of 10 microg/ml, after 24 h; while the level in control plasma was below detection limit. OMV-LPS did not induce release of IL-4 and IL-13 in doses from 0.001-10 microg/ml. The present results show that OMVs from meningococci have potent pro-inflammatory properties and are likely to contribute to the observed local and systemic inflammatory effects.     

Effects of Citrus unshiu powder on the cytokine balance in peripheral blood mononuclear cells of patients with seasonal allergic rhinitis to pollen

We evaluated the effects of a 50% methanol extract of Citrus unshiu powder (MEC) on cytokines in peripheral blood mononuclear cells (PBMCs) obtained from patients with seasonal allergic rhinitis to cedar pollen. The levels of cytokines, such as TNF-alpha, IFN-gamma, IL-2, IL-4, IL-5, IL-10, IL-12 (p70), IL-13, and GM-CSF, produced by pollen-stimulated PBMC were measured. We found that MEC suppressed pollen-induced TNF-alpha release and increased IFN-gamma release from PBMCs. The results suggest that Citrus unshiu powder has an immunomodulatory effect in vitro and that its use could improve seasonal allergic rhinitis symptoms.     

Transfer of severe experimental autoimmune encephalomyelitis by IL-12- and IL-18-potentiated T cells is estrogen sensitive

The aim of this study was to evaluate the roles of IL-18 and IL-12 in potentiating the encephalitogenic activity of T cell lines specific for myelin oligodendrocyte glycoprotein (MOG(35-55)). MOG-specific T cells stimulated with anti-CD3 and anti-CD28 in the presence of IL-12 or IL-18 alone transferred only mild experimental autoimmune encephalomyelitis (EAE) into a low percentage of recipients. However, T cells cocultured with both cytokines transferred aggressive clinical and histological EAE into all recipients. Coculture of T cells with IL-12 enhanced the secretion of IFN-gamma, but not TNF-alpha, whereas coculture with IL-18 enhanced the secretion of TNF-alpha, but not INF-gamma. However, coculture with both IL-18 and IL-12 induced high levels of both TNF-alpha and IFN-gamma. Additionally, IL-12 selectively enhanced mRNA expression of CCR5, whereas IL-18 selectively enhanced the expression of CCR4 and CCR7, and CCR4 and CCR5 were coexpressed on the surface of T cells cocultured with IL-12 and IL-18. Finally, estrogen treatment, previously found to inhibit both TNF-alpha and IFN-gamma production, completely abrogated all signs of passive EAE. These data demonstrate that optimal potentiation of encephalitogenic activity can be achieved by conditioning MOG-specific T cells with the combination of IL-12 and IL-18, which, respectively, induce the secretion of IFN-gamma/CCR5 and TNF-alpha/CCR4/CCR7, and that estrogen treatment, which is known to inhibit both proinflammatory cytokines, can completely ablate this aggressive form of passive EAE.     

High levels of IL-17 in rheumatoid arthritis patients: IL-15 triggers in vitro IL-17 production via cyclosporin A-sensitive mechanism

Recent data suggest that IL-15 plays an important role in the pathogenesis of rheumatoid arthritis. In the present study, we hypothesized that elevated in the joints of rheumatoid arthritis, but not osteoarthritis, patients, IL-15 may exert its proinflammatory properties via the induction of IL-17, a cytokine known to stimulate synoviocytes to release several mediators of inflammation including IL-6, IL-8, GM-CSF and PGE2. To test this hypothesis, we first measured the levels of IL-17 and IL-15 using specific ELISA and found that synovial fluids of patients with rheumatoid arthritis, but not with osteoarthritis, contain high levels of these cytokines. A strong correlation between IL-15 and IL-17 levels in synovial fluids was observed. Among tested factors, LPS and TNF-alpha failed, IL-15 and IL-2 were equipotent, and PMA + ionomycin was far more efficient in the induction of IL-17 secretion by PBMCs isolated from healthy blood donors. Interestingly, synovial fluid cells, in contrast to PBMCs isolated from patients with rheumatoid arthritis, but not osteoarthritis, respond to PMA + ionomycin with much lower, comparable to IL-15-triggered IL-17 secretion. Moreover, PMA + ionomycin-triggered IL-17 secretion is completely or partially blocked in the presence of low doses of cyclosporin A or high doses of methylprednisolone, respectively. IL-15-triggered IL-17 secretion by PBMCs was completely inhibited by these drugs. Thus, our results suggest for the first time that IL-15 may represent a physiological trigger that via cyclosporin A and steroid sensitive pathways leads to the overproduction of IL-17 in the joints of rheumatoid arthritis patients.     

Inhibition of IL-6, TNF-alpha, and cyclooxygenase-2 protein expression by prostaglandin E2-induced IL-10 in bone marrow-derived dendritic cells

Several endogenously produced mediators, including cytokines such as IL-6, IL-10, and TNF-alpha and prostanoids such as prostaglandin E(2) (PGE(2)), regulate dendritic cell (DC) function and contribute to immune homeostasis. In this study, we report that exogenous PGE(2) enhances the production of IL-10 from bone marrow-derived DC (BM-DC). IL-6, but not TNF-alpha, release is enhanced by PGE(2) in the presence of anti-IL-10, suggesting that endogenous IL-10 masks PGE(2)-induced IL-6. Furthermore, both exogenous IL-10 and PGE(2) inhibit LPS-induced IL-6 and TNF-alpha, whereas selective inhibition of cyclooxygenase-2 (COX-2) or addition of anti-IL-10 causes the reverse effects. Exogenous IL-10, but not IL-6, dose-dependently suppresses COX-2 protein expression and PGE(2) production, and TNF-alpha does not reverse this effect. In contrast, anti-IL-10 up-regulates prostanoid production by LPS-stimulated BM-DC. Taken together, our results show that in response to PGE(2), BM-DC produce IL-10, which in turn down-regulates their own production of IL-6-, TNF-alpha-, and COX-2-derived prostanoids, and plays crucial roles in determining the BM-DC pro-inflammatory phenotype.     

Direct influences of pro-inflammatory cytokines (IL-1beta, TNF-alpha, IL-6) on the proliferation and cell-surface antigen expression of cancer cells

Pro-inflammatory cytokines, e.g. interleukin 1 (IL-1), tumour necrosis factor alpha (TNF-alpha), IL-6 produced by surgical intervention or non-specific immunotherapy may directly affect both the growth and the metastasis of tumour cells. It is therefore important to clarify the direct influence of pro-inflammatory cytokines on tumour cells in order to obtain a better knowledge of anti-tumour therapy. Four human lung cancer cell lines were used. The tumour cells were incubated for 72 h in the presence of various concentrations of IL-1beta, TNF-alpha, or IL-6 and then the proliferative response was assessed by an MTT assay. After 14 days of culture with each pro-inflammatory cytokine, the cell-surface antigen expressions (HLA-class I, HLA-class II, CEA, sialyl Lewis(x)) were assessed by an immunocytochemical staining method. Among the various combinations of tumour cells (PC-9, PC-12, QG-56, QG-95) and cytokines (IL-1beta, TNF-alpha, IL-6), only TNF-alpha significantly exhibited an antiproliferative effect against PC-9 cells. However, various modulations of the cell-surface antigen expression by the cytokines were observed. The HLA-class I antigen expression of PC-9 was augmented by either TNF-alpha or IL-1beta. Furthermore, IL-1beta was able to induce CEA in PC-9, QG-56, and QG-95 cells while TNF-alpha was able to enhance the expression of sialyl Lewis(x)in QG-95 cells. Although the influence of pro-inflammatory cytokines on the growth of tumour cells was only slight, some modulations of the cell-surface antigen expression were notable. The augmentation of HLA-class I expression can thus improve the immunogenicity of tumour cells while the induction of CEA or sialyl Lewis(x)may therefore be associated with the promotion of metastasis.     

High-dose leptin activates human leukocytes via receptor expression on monocytes

Leptin is capable of modulating the immune response. Proinflammatory cytokines induce leptin production, and we now demonstrate that leptin can directly activate the inflammatory response. RNA expression for the leptin receptor (Ob-R) was detectable in human PBMCs. Ob-R expression was examined at the protein level by whole blood flow cytometry using an anti-human Ob-R mAb 9F8. The percentage of cells expressing leptin receptor was 25 +/- 5% for monocytes, 12 +/- 4% for neutrophils, and 5 +/- 1% for lymphocytes (only B lymphocytes). Incubation of resting PBMCs with leptin induced rapid expression of TNF-alpha and IL-6 mRNA and a dose-dependent production of TNF-alpha and IL-6 by monocytes. Incubation of resting PBMCs with high-dose leptin (250 ng/ml, 3-5 days) induced proliferation of resting cultured PBMCs and their secretion of TNF-alpha (5-fold), IL-6 (19-fold), and IFN-gamma (2.5-fold), but had no effect on IL-4 secretion. The effect of leptin was distinct from, and additive to, that seen after exposure to endotoxin or activation by the mixed lymphocyte reaction. In conclusion, Ob-R is expressed on human circulating leukocytes, predominantly on monocytes. At high doses, leptin induces proinflammatory cytokine production by resting human PBMCs and augments the release of these cytokines from activated PBMCs in a pattern compatible with the induction of Th1 cytokines. These results demonstrate that leptin has a direct effect on the generation of an inflammatory response. This is of relevance when considering leptin therapy and may partly explain the relationship among leptin, proinflammatory cytokines, insulin resistance, and obesity.     

Corticosteroids and interferons inhibit cytokine-induced production of IL-8 by human endothelial cells

IL-8, secreted by endothelial cells at the site of inflammation, participates in recruitment and transmigration of leukocytes. IL-8 may also have pathophysiological consequences in inflammatory and immunological disorders. We have investigated the effect of interferons (IFNs) and glucocorticosteroids (GCs) on cytokine induced secretion and production of IL-8 by human umbilical endothelial cells (HUVEC). There was a low spontaneous secretion of IL-8 by unstimulated HUVEC which increased after 6 or 24 h of stimulation with the pro-inflammatory cytokines TNF-alpha or IL-1beta. IFN-gamma as well as the GCs, Dexamethasone and Budesonide, inhibited TNF-alpha induced IL-8 secretion in a dose-dependent manner. IFNs may have a general modulating effect, since IFN-alpha also inhibited the TNF-alpha-induced IL-8 secretion. There was a slight, but significant, increase in the content of intracellular IL-8 in stimulated HUVEC. However, there was no difference between stimulation with IL-1beta or TNF-alpha alone or in combination with IFNs or GCs, whereas inhibition of IL-8 secretion with monensin increased IL-8 content suggesting that IFNs and GCs inhibit synthesis rather than secretion of IL-8. In conclusion, IFNs or GCs may be useful for inhibiting IL-8 production by endothelial cells and could thus be used for therapeutic modulation of the inflammatory response.     

Human intestinal mast cells are capable of producing different cytokine profiles: role of IgE receptor cross-linking and IL-4

Mast cells are recognized as a new type of immunoregulatory cells capable of producing different cytokines. So far, little is known about the cytokine profile of mature human mast cells isolated from intestinal tissue and cultured in the presence of stem cell factor (SCF). We observed that these cells express the proinflammatory cytokines TNF-alpha, IL-1 beta, IL-6, IL-8, IL-16, and IL-18 without further stimulation. Both IgE-dependent and IgE-independent agonists (e.g., Gram-negative bacteria) enhanced expression of TNF-alpha. Another set of cytokines consisting of IL-3, IL-5, IL-9, and IL-13 was expressed following activation by IgE receptor cross-linking. If mast cells were cultured in the presence of IL-4 and SCF, the production and release of IL-3, IL-5, and IL-13 was increased up to 4-fold compared with mast cells cultured with SCF alone. By contrast, IL-6 expression was completely blocked in response to culture with IL-4. In summary, our data show that mature human mast cells produce proinflammatory cytokines that may be up-regulated following triggering with IgE-independent agonists such as bacteria, whereas activation by IgE receptor cross-linking results in the expression of Th2-type cytokines. IL-4 enhances the expression of Th2-type cytokines but does not affect or even down-regulates proinflammatory cytokines.     

Differential effect of TPA on PGE2 and cicaprost-induced cAMP synthesis in UMR-106 cells.

PGE2 and prostacyclin each enhance cAMP synthesis in the osteoblast-like cell line UMR-106. The amount of cAMP induced by PGE2 was 5-7-fold greater than the amount induced by cicaprost or iloprost, stable prostacyclin analogues. Both PGE2 and the two prostacyclin analogues enhanced cAMP synthesis with similar time dependence. The EC50 values of PGE2 and cicaprost were 3 X 10(-6) and 5 x 10(-8) M, respectively. Short-term incubation of the cells with 12-o-tetradecanoylphorbol 13-acetate (TPA) markedly reduced the PGE2-induced cAMP synthesis. In contrast, cells that were incubated with the same concentrations of TPA in the presence of cicaprost or iloprost showed a 1.6-fold increase in cAMP formation. The marked disparity between the cAMP response to cicaprost and PGE2 in the presence of TPA suggests that the two prostanoids induce cAMP synthesis in the UMR-106 cells by interaction with different receptors. These observations support the idea that the osteoblastic UMR-106 cells may express specific prostacyclin receptors and suggest that prostacyclin may have a unique role in osteoblasts.     

IL-13 and IL-4 promote TARC release in human airway smooth muscle cells: role of IL-4 receptor genotype

The chemokine thymus- and activation-regulated chemokine (TARC) induces selective migration of Th2, but not Th1, lymphocytes and is upregulated in the airways of asthmatic patients. The purpose of this study was to determine whether human airway smooth muscle (HASM) cells produce TARC. Neither IL-4, IL-13, IL-1beta, IFN-gamma, nor TNF-alpha alone stimulated TARC release into the supernatant of cultured HASM cells. However, both IL-4 and IL-13 increased TARC protein and mRNA expression when administered in combination with TNF-alpha but not IL-1beta or IFN-gamma. Macrophage-derived chemokine was not expressed under any of these conditions. TARC release induced by TNF-alpha + IL-13 or TNF-alpha + IL-4 was inhibited by the beta-agonist isoproterenol and by other agents that activate protein kinase A, but not by dexamethasone. To determine whether polymorphisms of the IL-4Ralpha have an impact on the ability of IL-13 or IL-4 to induce TARC release, HASM cells from multiple donors were genotyped for the Ile50Val, Ser478Pro, and Gln551Arg polymorphisms of the IL-4Ralpha. Our data indicate that cells expressing the Val50/Pro478/Arg551 haplotype had significantly greater IL-13- or IL-4-induced TARC release than cells with other IL-4Ralpha genotypes. These data indicate that Th2 cytokines enhance TARC expression in HASM cells in an IL-4Ralpha genotype-dependent fashion and suggest that airway smooth muscle cells participate in a positive feedback loop that promotes the recruitment of Th2 cells into asthmatic airways.     

The effects of some Curdlan derivatives on Dectin-1 expression and cytokine production in human peripheral blood mononuclear cells

The cells of immune system such as monocytes and macrophages are in first line defence against dangerous signals. In the present paper the recognition of Dectin 1 receptors and the modulation of Interleukin-10 (IL-10) and Tumor Necrosis Factor-alpha (TNF-alpha) cytokine production by Curdlan and Curdlan derivatives in peripheral blood mononuclear cells (PBMCs) were studied. The effect of Curdlan or Curdlan derivatives on the expression of Dectin 1 receptors in PBMCs was revealed by flow-cytometry and the levels of IL-10 and TNFalpha were measured by ELISA kit in supernatants of PBMCs cultured in presence or absence of Curdlan, Curdlan derivatives and LPS. Our results suggested that Curdlan and Curdlan derivatives were able to increase the expression of Dectin-1 receptors on monocyte cells. The combined treatment of Curdlan/Curdlan derivatives and Pam3Cys produced an increase of CD14+ cells possessing Dectin-1 receptors. We demonstrated that Curdlan (at 20 microg unique dose) up-regulated TNF-alpha production and down-regulated IL-10 production in PBMCs. Conversely, Palm CM/SP-Curdlan (20 microg unique dose) was able to down-regulate TNF-alpha production and to up-regulate IL-10 production in PBMCs. For instance, Palm CM/SP-Curdlan determined a 5 times decrease of TNF-alpha production than Curdlan. Regarding the effect of Palm CM/SP-Curdlan on IL-10 production in PBMCs, we noticed that the level of IL-10 was about 4 times greater than Curdlan activity. We observed that a combined treatment of Curdlan/Curdlan derivatives and LPS induced about 5 times decrease in TNF-alpha production in PBMCs. IL-10 production induced by Palm CM/SP-Curdlan and LPS was about 6 times greater than the combined effect of Curdlan and LPS. The treatment of PBMCs with SP-Curdlan alone affected neither TNF-alpha production nor IL-10 production. Our results are in accordance with other studies demonstrating that Dectin-1 and TLR2/TLR6 signaling combine to enhance the responses triggered by each receptor and the signaling pathway induced by Dectin-1 could mediate the production of pro-inflammatory cytokines.     

Cytokine-mediated modulation of leptin and adiponectin secretion during in vitro adipogenesis: evidence that tumor necrosis factor-alpha- and interleukin-1beta-treated human preadipocytes are potent leptin producers

Over the last decade, compelling evidence has been presented that cytokines affect adipocyte tissue formation and function. In this study we explored the effect of pro-inflammatory (i.e. interleukin (IL)-1beta, IL-6, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha) versus anti-inflammatory cytokines (i.e. IL-4, IL-10, and transforming growth factor (TGF)-beta1) on leptin and adiponectin secretion during in vitro human adipogenesis. Confirmative to previous reports, conversion of precursor preadipocytes into mature adipocytes was completely inhibited upon exposure to TNF-alpha, IL-1beta, IFN-gamma, or TGF-beta1. Hence, all these anti-adipogenic cytokines prevented release of adipocyte-specific adiponectin. IFN-gamma also strongly reduced leptin production (> or =85%). However, TNF-alpha, IL-1beta, and TGF-beta1 stimulated leptin production from preadipocytes in the absence of mature adipocytes (20.6+/-5.4 ng/ml, 100.8+/-18.2 ng/ml, and 5.4+/-0.4 ng/ml, respectively, compared to 6.6+/-0.8 ng/ml in control adipocyte cultures on day 21; n=4). IL-4, IL-6 and IL-10 did not, or only slightly, affect adipocyte differentiation and their hormonal secretion. In conclusion, adiponectin and leptin are both synthesized by adipocytes, whereas leptin is also produced by preadipocytes upon TNF-alpha or IL-1beta stimulation. These data suggest that preadipocytes could contribute more to total circulating leptin levels than has been previously considered, especially in diseased conditions were these pro-inflammatory factors play a prominent role.     

Toll-like receptor (TLR) 2-9 agonists-induced cytokines and chemokines: I. Comparison with T cell receptor-induced responses

The cells of innate and adaptive immunity, although activated by different ligands, engage in cross talk to ensure a successful immune outcome. To better understand this interaction, we examined the demographic picture of individual TLR (TLRs 2-9) -driven profiles of eleven cytokines (IFN-alpha/beta, IFN-gamma, IL-12p40/IL-12p70, IL-4, 1L-13, TNF-alpha, IL-1beta, IL-2, IL-10) and four chemokines (MCP-1, MIP1beta, IL-8, and RANTES), and compared them with direct T-cell receptor triggered responses in an assay platform using human PBMCs. We find that T-cell activation by a combination of anti-CD3/anti-CD28/PHA induced a dominant IL-2, IL-13, and Type-II interferon (IFN-gamma) response without major IL-12 and little Type-I interferon (IFN-alphabeta) release. In contrast, TLR7 and TLR9 agonists induced high levels of Type-I interferons. The highest IFN-gamma levels were displayed by TLR8 and TLR7/8 agonists, which also induced the highest levels of pro-inflammatory cytokines IL-12, TNF-alpha, and IL-1beta. Amongst endosomal TLRs, TLR7 displayed a unique profile producing weak IL-12, IFN-gamma, TNF-alpha, IL-1beta, and IL-8. TLR7 and TLR9 resembled each other in their cytokine profile but differed in MIP-1beta and MCP1 chemokine profiles. Gram positive (TLR2, TLR2/6) and gram negative (TLR4) pathogen-derived TLR agonists displayed significant similarities in profile, but not in potency. TLR5 and TLR2/6 agonists paralleled TLR2 and TLR4 in generating pro-inflammatory chemokines MCP-1, MIP-1beta, RANTES, and IL-8 but yielded weak TNF-alpha and IL-1 responses. Taken together, the data show that diverse TLR agonists, despite their operation through common pathways induce distinct cytokine/chemokine profiles that in turn have little or no overlap with TCR-mediated response.     

Mitogenic action of tumour necrosis factor-alpha and interleukin-8 on explants of human duodenal mucosa

Our aim is to examine whether tumour necrosis factor-alpha (TNF-alpha) and interleukin affect the mitotic activity in explants of human duodenal mucosa and to estimate the release of cytokines from explants incubated with TNF-alpha. Biopsy specimens of normal duodenal mucosa were taken from 19 subjects that underwent upper endoscopy for investigation of dyspeptic symptoms or chronic gastrointestinal bleeding. The specimens were processed following guidelines for organ culture technique. Paired biopsy specimens from 12 subjects were cultured for 23 h to achieve steady state and thereafter the explants were incubated 25 h with 10(-13)-10(-9) M of TNF-alpha or IL-8. Mitoses were arrested in the metaphase by adding vincristine sulphate for the last three hours. The explants were then fixed and processed for microdissection. Fifteen crypts were microdissected and the total number of metaphases was determined using the whole crypt as reference volume. The number of metaphases per crypt was also estimated in explants incubated with 10(-10) M TNF-alpha in the presence of anti-IL-8 antibodies. Additional duodenal explants from seven subjects were incubated with 10(-10) M TNF-alpha for 25 h. Thereafter the release of IL-1-beta, IL-6, IL-8 and interferon gamma (IFN-gamma) into the culture medium was measured by enzyme immunoassay and expressed as pg/mg protein. TNF-alpha and IL-8 significantly increased the number of metaphases/crypts (P<0.0001). The addition of anti-IL-8 slightly reduced the number of metaphases/crypt compared to the values observed in the explants incubated with 10(-10) M TNF-alpha alone (P<0.0001). The number of metaphases/crypt in the explants incubated with 10(-10) M TNF-alpha in the presence of anti-IL-8 antibodies was, however, markedly and significantly higher than that of the controls (P<0.000). TNF-alpha induced the release of IL-8 (P<0.01) and IL-6 (P<0.05) from the duodenal explants. TNF-alpha and IL-8 are potent mitogens to human small intestinal crypts. The mitogenic action of TNF-alpha is primarily a direct effect of the cytokine and only to a minor extent mediated by a secondary production of IL-8 in the duodenal explant. Our findings indicate that TNF-alpha and IL-8 may participate in the regulation of cell proliferation in the human small intestinal epithelium.     

Regulation of IL-15-stimulated TNF-alpha production by rolipram.

Agents that increase intracellular cAMP have been shown to reduce joint inflammation in experimental arthritis, presumably by lowering the release of proinflammatory cytokines, such as TNF-alpha. Recent studies suggest that, in joints of patients with rheumatoid arthritis, TNF-alpha release from macrophages is triggered by their interaction with IL-15-stimulated T lymphocytes. In this report, we analyze the effect of rolipram, a cAMP-specific phosphodiesterase inhibitor, on TNF-alpha production in this experimental system. Cocultures of U937 cells with IL-15-stimulated T cells, but not control T cells, resulted in increased release of TNF-alpha. Pretreatment of T cells with rolipram or cAMP analogues inhibited the IL-15-stimulated increases in proliferation, expression of cell surface molecules CD69, ICAM-1, and LFA-1, and release of TNF-alpha from macrophages. Addition of PMA to T cells dramatically increased the expression of cell surface molecules, but had little or no effect on TNF-alpha release from either T cells or from cocultures, suggesting that other surface molecules must also be involved in T cell/macrophage contact-mediated production of TNF-alpha. Addition of PMA synergistically increased the proliferation of IL-15-stimulated T cells and the secretion of TNF-alpha from IL-15-stimulated T cell/macrophage cocultures. Rolipram and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) blocked these increases. Measurement of protein kinase A (PKA) activity and the use of inhibitory cAMP analogues (RpCPT-cAMP) confirmed that rolipram worked by stimulating PKA. These data suggest that PKA-activating agents, such as rolipram, can block secretion of TNF-alpha from macrophages by inhibiting T cell activation and expression of surface molecules.