Neutrophil serine proteinases activate human nonepithelial cells to produce inflammatory cytokines through protease-activated receptor 2

Protease-activated receptors (PARs) compose a family of G protein-coupled receptors activated by proteolysis with exposure of their tethered ligand. Recently, we reported that a neutrophil-derived serine proteinase, proteinase 3 (PR3), activated human oral epithelial cells through PAR-2. The present study examined whether other neutrophil serine proteinases, human leukocyte elastase (HLE), and cathepsin G (Cat G) activate nonepithelial cells, human gingival fibroblasts (HGF). HLE and Cat G as well as PR3 activated HGF to produce IL-8 and monocyte chemoattractant protein 1. Human oral epithelial cells but not HGF express mRNA and protein of secretory leukocyte protease inhibitor, an inhibitor of HLE and Cat G, and recombinant secretory leukocyte protease inhibitor clearly inhibited the activation of HGF induced by HLE and Cat G but not by PR3. HGF express PAR-1 and PAR-2 mRNA in the cells and the proteins on the cell surface. HLE and Cat G cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca(2+) mobilization and rendered cells refractory to subsequent stimulation with HLE and Cat G. The production of cytokine induced by HLE and Cat G and the PAR-2 agonist peptide was completely abolished by inhibition of phospholipase C. These findings suggest that neutrophil serine proteinases have equal ability to activate human nonepithelial cells through PAR-2 to produce inflammatory cytokines and may control a number of inflammatory processes such as periodontitis.     

Membrane proteases: roles in tissue remodeling and tumour invasion.

The coordinated control of extracellular matrix degradation on the cell surface involves three crucial elements: secreted proteases and their inhibitors, surface protease receptors and integral membrane proteases. The roles that each of these elements play in cell surface proteolysis are described. The localization of proteases to the cell surface, protease activation, and regulation of cell surface proteolysis by protease inhibitors are key issues for elucidating the role of membrane proteases in tissue remodeling and tumour invasion.     

Proteases produced by activated neutrophils are able to release soluble CD23 fragments endowed with proinflammatory effects.

Polymorphonuclear neutrophils (PMNs) are the major source of proteolytic activities involved mainly in tissue injuries observed in chronic inflammatory disorders. High levels of soluble forms of CD23 (the low-affinity receptor for IgE) were found in biological fluids from these patients, and recent reports focused on a CD23-mediated regulation of inflammatory response. In this context, we show here that co-culture of activated PMN with CD23+ B cells resulted in a drastic release of soluble CD23 fragments from the cell surface. This cleavage was inhibited by serine proteases inhibitors, including a1-antitrypsin. We next demonstrated that purified human leukocyte elastase or cathepsin G efficiently cleaved membrane CD23 on B cells with a high specificity. Soluble fragments released by serine proteases-mediated CD23 proteolysis stimulated resting monocytes to produce oxidative burst and proinflammatory cytokine without any co-stimulatory signal. This work strongly supports the idea that the capacity of PMN-derived proteases to release soluble forms of CD23 participates in the inflammatory process mediated by these cells.     

Production of human mutant biologically active hepatocyte growth factor in Chinese hamster ovary cells

Hepatocyte growth factor (HGF) is a potent multifunctional cytokine that affects proliferation, migration, and morphogenesis of various cells. HGF is secreted as an inactive single-chain precursor protein and activated by the cleavage of serine proteases to form heterodimers. In our current study, the cleavage site of HGF was blocked by replaced Arg 494 of Glu (R494E) that resulted in the single-chain HGF (R494E) unable to be cleaved by serine proteases. We established Chinese hamster ovary (CHO) cells overexpressing HGF (R494E), the expression of HGF (R494E) achieved 12?mg/L and was similar to a previously reported study. The recombinant protein was then purified from culture medium using a two-step chromatographic procedure that resulted in about a 40% recovery rate. The purified HGF (R494E) was obtained as a single-chain active protein. It concluded that HGF (R494E) exhibited a biologically active protein and the overexpressing CHO cell line supplied sufficient material for future studies. The R494E replacement of the cleavage site would be beneficial to the utility of other similar therapeutic proteins.     

Suppression of human leukocyte tumor necrosis factor secretion by the serine protease inhibitor p-toluenesulfonyl-L-arginine methyl ester (TAME)

The addition of the serine protease inhibitor p-toluenesulfonyl-L-arginine methyl ester (TAME) to human peripheral blood mononuclear cells suppressed TNF secretion in a concentration dependent manner. At a concentration 10 mM TAME leukocyte TNF release was completely inhibited without decreasing the secretion of IL-1 alpha. Simultaneously exposing leukocytes to 10 mM TAME and either 1000 U/ml IFN-gamma or 10 micrograms/ml LPS reduced the quantity of TNF secreted by 75% and 47%, respectively, when compared with the effect of either IFN-gamma or LPS alone. TAME was most effective when added to leukocytes at the initiation of culture and the suppressive effects of this protease inhibitor were reversible by washing the cells. TAME suppressed TNF secretion without affecting either the level of TNF mRNA or the expression of cell surface cytokine. These findings suggest that leukocyte TNF secretion is dependent upon the action of one or more serine proteases.     

Cleavage of CD14 and LBP by a protease from<Emphasis Type="Italic"> Prevotella intermedia</Emphasis>

Periodontitis is an inflammatory disease caused by subgingival microorganisms and their components, such as lipopolysaccharide (LPS). Responses of the host to LPS are mediated by CD14 and LPS-binding protein (LBP). In this study, it was determined that proteases from a periodontal pathogen, Prevotella intermedia, cleave CD14 and LBP, and thereby modulate the virulence of LPS. Culture supernatants from two strains of P. intermedia (ATCC 25611 and 25261) cleaved CD14 and LBP in a concentration-dependent manner. Zymographic and molecular mass analysis revealed the presence of a membrane-associated, 170-kDa, monomeric protease. Class-specific inhibitors and stimulators demonstrated that this enzyme is a metal-requiring, thiol-activated, cysteine protease. The protease was stable over a wide range of temperatures (4-56 degrees C) and pH values (4.5-8.5). This enzyme also decreased the expression of interleukin-1beta (IL-1beta)-specific mRNA in the LPS-activated macrophage-like cell lines U937 and THP-1 in a concentration-dependent manner, indicating that it also cleaves membrane-associated CD14. Furthermore, addition of soluble CD14 abrogated protease-mediated inhibition of IL-1 mRNA expression induced by LPS. The observations suggest that proteolysis of CD14 and LBP by P. intermedia protease might modulate the virulence of LPS at sites of periodontal infections.     

Analysis of the mechanisms of T cell-dependent polyclonal activation of human B cells. Induction of human B cell responses by fixed activated T cells.

Coculture of resting human B cells with T cells stimulated with immobilized mAb to the CD3 molecular complex induces polyclonal activation and the production of Ig of all isotypes. The current experiments were carried out to determine the nature of the signals provided to B cells by the anti-CD3-activated T cells. For these experiments, fresh T cells or T cell clones were activated with immobilized mAb to CD3 and then fixed with 1% paraformaldehyde. Upon coculture, the fixed activated T cells or T cell clones induced B cell RNA synthesis and IL-2R expression, but only minimal DNA synthesis and no Ig production. Induction of B cell RNA synthesis by fixed activated T cells was not inhibited by mAb to the alpha-chain of the IL-2R, and was not enhanced by supplementing cultures with IL-2, IL-4, IL-6, or supernatants of mitogen-activated T cells. Upon the addition of IL-2, but not IL-4 or IL-6, to cultures of B cells and fixed activated T cells, sustained proliferation was noted along with the production of Ig. Control fixed T cells or T cell clones did not induce any of these responses. The presence of cycloheximide or cyclosporin A during the activation with anti-CD3 prevented T cells from developing the capacity to provide help for B cells. The use of mAb to a variety of cell surface molecules indicated that several T cell surface molecules including CD11a/CD18, CD44, CD54, and class I MHC molecules are involved in the induction of B cell responses. Among the mAb that inhibited B cell DNA synthesis and/or Ig production, only mAb to CD11a, CD18, or CD54 inhibited initial B cell activation as assessed by RNA synthesis. Even though mAB to CD11a/CD18 inhibited the capacity of fixed activated T cells to induce B cell responses, the finding that fixed activated CD18 deficit clones provided help for B cells indicated that expression of the beta 2 family of integrins by T cells was not necessary. These results indicate that activated T cells acquire the capacity to stimulate B cells polyclonally and induce cytokine responsiveness, proliferation, and Ig production by utilization of a variety of surface molecules. Moreover, these results indicate that the initial activation of the B cell is independent of the metabolic activity of the T cell and the production of cytokines.     

Possible role of calpain in normal processing of beta-amyloid precursor protein in human platelets

Abnormal proteolytic processing of beta-amyloid precursor protein (APP) underlies the formation of amyloid plaques in aging and Alzheimer's disease. The proteases involved in the process have not been identified. Here we found that spontaneous proteolysis of intact APP in detergent-lysed human platelets generated a N-terminal fragment that was immunologically indistinguishable from secreted APP, reminiscent of the action of a putative alpha-secretase. This proteolysis of APP was inhibited by EDTA, suggesting that a metal-dependent protease was involved. Among the several metals tested, calcium was the only one that enhanced APP proteolysis and the reaction was blocked by EGTA as well as by several calpain inhibitors. The APP fragments generated by spontaneous proteolysis in platelet lysates were identical to those produced by exposure of partially purified APP to exogenous calpain. Finally, the secretion of APP from intact platelets was inhibited by cell-permeable calpain inhibitors. Taken together, these results suggest that normal processing of APP in human platelets is mediated by a calcium-dependent protease that exhibits calpain-like properties.     

Role of cathepsin D in antigen presentation of ovalbumin.

Modification of protein Ag by proteolysis is one of the principal steps in the presentation of Ag to Th cells. However, little is known about the enzymes participating in these events, their specificity or the characteristics of the natural fragments that they produce. Cathepsin D (CD) is an aspartyl protease identified in endosomes of APC. In this report, the role of CD in the processing of OVA has been investigated. OVA digested in vitro with purified CD was able to stimulate IL-2 secretion by three different OVA-specific I-Ad restricted Th cell hybridomas when it was presented by fixed APC. The digest of OVA was recognized in the context of I-Ad, but not by I-Ak-restricted OVA-specific Th cells. No difference was observed in the ability of OVA digested with CD to stimulate Th cells in the absence of FCS or in the presence of protease inhibitors indicating that extracellular proteases were not likely to contribute to processing of OVA. Taken together, these results suggest that CD is necessary and sufficient for the generation of an antigenic epitope from OVA. A fragment containing the epitope was isolated from the OVA digest by reverse phase HPLC. This fragment, which migrates in SDS-PAGE as a 10-kDa polypeptide, is a potent epitope. Its capacity to activate Th cells is compared to that of the tryptic peptide OVA323-339.     

Cultured human skeletal muscle satellite cells exhibit characteristics of mesenchymal stem cells and play anti-inflammatory roles through prostaglandin E2 and hepatocyte growth factors

Skeletal muscle satellite cells (SkMSCs) play crucial roles in muscle fiber maintenance, repair, and remodeling; however, it remains unknown if these properties are preserved in cultured SkMSCs. In this study, we investigated the characteristics of cultured SkMSCs and their ability to regulate the activity of M1 macrophages. SkMSCs grew well with an average population doubling time of 26.26 ± 6.85 h during 10 passages (P). At P5, Pax7, MyoD, cluster of differentiation (CD)34, and CD56 were not expressed in SkMSCs, but the MSC markers CD73, CD105, and CD90 were expressed and the cells were differentiated into adipocytes and osteoblasts. When SkMSCs were cocultured with macrophages, interleukin (IL)-1β secretion was decreased, prostaglandin (PG)E2 was produced in coculture, and cyclooxygenase-2 protein was induced in an SkMSC-dependent manner. Hepatocyte growth factor (HGF) was highly secreted by monocultured SkMSCs; interferon-γ and lipopolysaccharide reduced its expression level. However, HGF expression recovered when SkMSCs and macrophages were cocultured. Although exogenous PGE2 upregulated macrophage pro-IL-1β expression, it suppressed the secretion of cleaved IL-1β. In contrast, HGF decreased active IL-1β secretion without affecting pro-IL-1β expression. Co-treatment of macrophages with HGF and PGE2 reduced pro-IL-1β expression level and active IL-1β secretion. Our results suggest that SkMSCs lose their satellite cell properties during serial passaging but acquire mesenchymal stem cell properties including the ability to exert an anti-inflammatory response for macrophages through PGE2 and HGF.     

Basic fibroblast growth factor stimulates hepatocyte growth factor/scatter factor secretion by human mesenchymal cells

Basic fibroblast growth factor (bFGF) together with other pleiotropic factors plays an important role in many complex physiological processes such as embryonic development, angiogenesis, and wound repair. Among these factors, hepatocyte growth factor/scatter factor (HGF/SF) which is secreted by cells of mesodermal origin exerts its mito- and motogenic activities on cells of epithelial and endothelial origin. Knowledge of the regulatory mechanisms of HGF/SF may contribute to the understanding of its role in physio-pathological processes. We observed that the secretion of HGF/SF by MRC-5 cells and by other fibroblast-derived cell cultures in conditioned media was enhanced by exposure to bFGF. HGF/SF was measured by the scatter assay, a bioassay for cell motility, and was further characterized by Western blot analysis with anti-HGF/SF antibodies. Exposure of MRC-5 cultures to 10 ng/ml of bFGF resulted already 6 h posttreatment in a threefold higher amount of scatter factor secreted into the medium as compared to untreated cultures. HGF/SF secretion was sustained after bFGF treatment for the following 72 h when increased amounts of HGF/SF were detected both in conditioned media as well as associated to the extracellular matrix. The secretion of HGF/SF in cell supernatants increased dose dependently upon treatment with bFGF starting from basal levels of 6 U/ml and reaching 27 U/ml at 30 ng/ml bFGF, plateauing thereafter. Upregulation of HGF/SF by IL-1, already described by others, was confirmed in this study. Based on our findings an articulated interaction can be speculated for bFGF, HGF/SF, and IL-1, e.g., in tissue regeneration during inflammatory processes or in wound healing. © 1996 Wiley-Liss, Inc.     

Effects of glutamine on adhesion molecule expression and leukocyte transmigration in endothelial cells exposed to arsenic

This study evaluated whether glutamine (GLN) concentration was related to endothelial surface molecule expression and the migration of polymorphonuclear neutrophils (PMNs) through endothelial cells (ECs) stimulated by arsenic. Human umbilical vein endothelial cells (HUVECs) and PMNs were treated with different GLN concentrations (0, 300, 600 and 1000 microM) for 24 h. After that, we stimulated HUVECs for 3 h with 0.5 microM arsenic, and PMNs were allowed to transmigrate to ECs for 2 h. HUVEC surface expressions of cell adhesion molecules and integrin (CD11b) and interleukin (IL)-8 receptor expressions on PMNs were measured. The transendothelial migration of PMNs was also analyzed. The results showed that cell adhesion molecule (CAM) and integrin expressions in arsenic groups were higher than in those without arsenic. Among the arsenic groups, the expression of CAMs on ECs and CD11b, and IL-8 receptor on PMNs was lowest with 0 microM compared with the other GLN concentrations. Vascular CAM-1 on ECs and CD11b on PMN expression were higher with 300 microM than with 600 and 1000 microM GLN. IL-8 secretions from ECs and PMNs were higher with 300 muM than with 600 and 1000 microM GLN, and this was consistent with the expression of the IL-8 receptor on PMNs. Polymorphonuclear neutrophil transmigration was significantly higher with 300 muM GLN than with other GLN concentrations. These results suggest that ECs and PMNs were activated after arsenic stimulation. Cell adhesion molecule expressions on ECs and PMNs were suppressed in the absence of GLN. A low GLN concentration comparable to catabolic conditions resulted in higher adhesion molecule expression and greater transendothelial migration of neutrophils. Glutamine administration at levels similar to or higher than physiological concentrations reduced IL-8 and adhesion molecule expression; PMN transmigration was also decreased after stimulation with arsenic.     

The biosynthesis of endothelin-1 by human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMNs) converted human big endothelin (bET; 2 microM) to an endothelin-1 (ET-1) like contractile factor, as assessed by bioassay. The generation of this ET-1 like activity was rapid (minutes), time-dependent and more pronounced in non-activated cells, suggesting a partial degradation by activated PMNs. Phosphoramidon (54 micrograms/ml) inhibited the formation of this contractile factor, whereas phenylmethylsulfonylfluoride (PMSF; 25 micrograms/ml), pepstatin A (1 microgram/ml) or epoxysuccinyl-L-leucylamido-(guanidino)butane (E-64; 10 micrograms/ml) did not. Incubations of activated PMNs with PMSF significantly potentiated the generation of ET-1 like activity and selectively inhibited the degradation of [125I]ET-1 by activated PMNs. These findings indicate that human PMNs contain and/or release neutral proteases, which can both rapidly produce and degrade ET-1, an observation which may have important (patho)physiologic implications.     

A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes

The amiloride-sensitive epithelial sodium channel (ENaC) constitutes a limiting step in sodium reabsorption across distal airway epithelium and controlling mucociliary clearance. ENaC is activated by serine proteases secreted in the extracellular milieu. In cystic fibrosis lungs, high concentrations of secreted neutrophil elastase (NE) are observed. hNE could activate ENaC and contribute to further decreased mucociliary clearance. The aims of this study were (i) to test the ability of an engineered human neutrophil elastase inhibitor (EPI-hNE4) to specifically inhibit the elastase activation of ENaC-mediated amiloride-sensitive currents (I(Na)) and (ii) to examine the effect of elastase on cell surface expression of ENaC and its cleavage pattern (exogenous proteolysis). Oocytes were exposed to hNE (10-100 microg/ml) and/or trypsin (10 microg/ml) for 2-5 min in the presence or absence of EPI-hNE4 (0.7 microm). hNE activated I(Na) 3.6-fold (p < 0.001) relative to non-treated hENaC-injected oocytes. EPI-hNE4 fully inhibited hNE-activated I(Na) but had no effect on trypsin- or prostasin-activated I(Na). The co-activation of I(Na) by hNE and trypsin was not additive. Biotinylation experiments revealed that cell surface gamma ENaC (but not alpha or beta ENaC) exposed to hNE for 2 min was cleaved (as a 67-kDa fragment) and correlated with increased I(Na). The elastase-induced exogenous proteolysis pattern is distinct from the endogenous proteolysis pattern induced upon preferential assembly, suggesting a causal relationship between gamma ENaC cleavage and ENaC activation, taking place at the plasma membrane.     

Cis- and trans-diamminedichloroplatinum(II) binding products different tertiary structural changes on SV40 DNA

The proteases secreted into culture medium by MCF-7 breast cancer cells produced both plasminogen-dependent and -independent proteolysis, as shown by casein-polyacrylamide gel electrophoresis. All of these proteases except the largest (M_r 120,000) were retained on a benzamidine-Sepharose affinity column, a characteristic of trypsinlike proteases. Among the proteases which activated plasminogen, all except a major protease of M_r 59,000 were antigenically similar to urokinase. These urokinaselike proteases (M_r 65,000 to 25,000) were isolated on a antiurokinase-Sepharose affinity column. The findings indicate that in a stable cell line derived from a human breast cancer there are two distinct types of plasminogen activators, opening the possibility that these activator types may be modulated in separate ways.     

Interleukin-18 enhances HIV-1 production in a human chronically-infected T cell line (H9-V)

Interleukin-18 (IL-18) is a recently identified immunoregulatory cytokine expressed by activated macrophages, that induces production of interferon-gamma (IFN-gamma) and Th-1 development. Recently some investigators reported controversial in vitro data on IL-18 stimulation of HIV-1 replication in several cell lines. In the present study the effect of IL-18 on HIV replication in a human chronically HIV-1-infected lymphocytic T cell line (H9-V) was investigated. HIV-1 replication was determined by an immunoassay method in order to evaluate the content of p24 antigen in the cell culture supernatants. Stimulation of H9-V cells with IL-18 resulted in increased production of p24, especially at concentrations of 0.01 microg ml(-1) and 0.10 microg ml(-1). Moreover a significant and persistent IL-18 stimulation of HIV-1 replication was observed at a concentration of 0.01 microg ml(-1) during a 7-day period. Pre-treatment of IL-18 with a specific neutralizing monoclonal antibody significantly reduced HIV-1 replication. These experiments show that IL-18 promotes the increase of HIV-1 replication in human chronically-infected lymphocytic T cells and confirm the role of IL-18 as a proimflammatory cytokine in stimulating and maintaining HIV-1 replication during the course of the disease. In a successive set of experiments, since one of the main activities of IL-18 is the induction of IFN-gamma, we evaluated the effect of this biological modifier on H9-V cells. In particular, IFN-gamma shows a significant effect on cell replication and on reduction of CD4 and CD71 surface expression.     

Generation of biologically active interleukin-1 beta by proteolytic cleavage of the inactive precursor

Interleukin-1 beta (IL-1 beta) is derived from an inactive precursor by proteolytic cleavage. To study IL-1 beta processing, we expressed the precursor in Escherichia coli, partially purified it, and used it as a substrate for various potentially relevant protease preparations. The precursor alone was virtually inactive, but incubation with membranes from human monocytes or myeloid cell lines yielded a 500-fold increase in IL-1 bioactivity. Western blot analysis of the incubated material showed that the 31,000-Da precursor is broken down to three major products, ranging from 17,400 to about 19,000 Da. The most active of these products is the smallest one, and it co-migrates during electrophoresis with mature IL-1 beta. Four purified known proteases were also tested for their effect on precursor IL-1 beta, and none of these products co-migrated with the mature protein. Chymotrypsin and Staphylococcus aureus protease yielded slightly larger products, which were highly active. Elastase and trypsin yielded substantially larger products, and these had little IL-1 activity. The products of three of the known proteases were identified by NH2-terminal sequencing. These results show conclusively that proteolysis of precursor IL-1 beta generates biological activity and that the cleavage must occur close to the mature NH2 terminus.     

Generation of biologically active interleukin-1beta by meprin B

Interleukin-1beta (IL-1beta) is a proinflammatory cytokine that is synthesized as an inactive precursor molecule that must be proteolytically processed to generate the biologically active form. Maturation of the precursor is primarily performed by caspase-1, an intracellular cysteine protease; however, processing by other proteases has been described. Meprins are cell surface and secreted metalloproteases expressed by renal and intestinal brush-border membranes, leukocytes, and cancer cells. In this study we show that purified recombinant meprin B can process the interleukin-1beta precursor to a biologically active form. Amino-terminal sequencing and mass spectrometry analysis of the product of digestion by activated meprin B determined that proteolytic cleavage resulted in an additional six amino acids relative to the site utilized by caspase-1. The biological activity of the meprin B-cleaved cytokine was confirmed by measuring the proliferative response of helper T-cells. These results suggest that meprin may play an important role in activation of this proinflammatory cytokine in various pathophysiological conditions.     

Recombinant expression of ShPI-1A, a non-specific BPTI-Kunitz-type inhibitor, and its protection effect on proteolytic degradation of recombinant human miniproinsulin expressed in Pichia pastoris

Pichia pastoris is a highly successful system for the large-scale expression of heterologous proteins, with the added capability of performing most eukaryotic post-translational modifications. However, this system has one significant disadvantage - frequent proteolytic degradation by P. pastoris proteases of heterologously expressed proteins. Several methods have been proposed to address this problem, but none has proven fully effective. We tested the effectiveness of a broad specificity protease inhibitor to control proteolysis. A recombinant variant of the BPTI-Kunitz protease inhibitor ShPI-1 isolated from the sea anemone Stichodactyla helianthus, was expressed in P. pastoris. The recombinant inhibitor (rShPI-1A), containing four additional amino acids (EAEA) at the N-terminus, was folded similarly to the natural inhibitor, as assessed by circular dichroism. rShPI-1A had broad protease specificity, inhibiting serine, aspartic, and cysteine proteases similarly to the natural inhibitor. rShPI-1A protected a model protein, recombinant human miniproinsulin (rhMPI), from proteolytic degradation during expression in P. pastoris. The addition of purified rShPI-1A at the beginning of the induction phase significantly protected rhMPI from proteolysis in culture broth. The results suggest that a broad specificity protease inhibitor such as rShPI-1A can be used to improve the yield of recombinant proteins secreted from P. pastoris.     

IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells.

Murine IL-10 has been reported originally to be produced by the Th2 subset of CD4+ T cell clones. In this study, we demonstrate that human IL-10 is produced by Th0, Th1-, and Th2-like CD4+ T cell clones after both Ag-specific and polyclonal activation. In purified peripheral blood T cells, low, but significant, levels of IL-10 were found to be produced by the CD4+CD45RA+ population, whereas CD4+CD45RA- "memory" cells secreted 5- to 20-fold higher levels of IL-10. In addition, IL-10 was produced by activated CD8+ peripheral blood T cells. Optimal induction of IL-10 was observed after activation by specific Ag and by the combination of anti-CD3 mAb and the phorbol ester tetradecanoyl phorbol acetate, whereas the combination of calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate acetate was a poor inducer of IL-10 production. Kinetic studies indicated that IL-10 was produced relatively late as compared with other cytokines. Maximal IL-10 mRNA expression in CD4+ T cell clones and purified peripheral blood T cells was obtained after 24 h, whereas maximal IL-10 protein synthesis occurred between 24 h and 48 h after activation. No differences were observed in the kinetics of IL-10 production among Th0, Th1-, and Th2-like subsets of CD4+ T cell clones. The results indicate a regulatory role for IL-10 in later phases of the immune response.