Dynamics of cytokine production in human peripheral blood mononuclear cells stimulated by LPS or infected by Borrelia

To specify the role of individual cytokines in the immune response to pyrogens, isolated and cultivated human peripheral blood mononuclear cells (PBMC) were used for the experiments. Different pyrogens (lipopolysaccharide from Escherichia coli - LPS and live Borrelia afzelii) were applied and the time course of changes in concentrations of different cytokines in the medium was followed using the ELISA method. It was found that nonstimulated human PBMC proliferate under in vitro conditions and produce IL-6, TNF-alpha, IL-10 and finally also IL-1beta. Productions of IL-12 and INF-gamma are not changed. Proliferation of PBMC is potentiated after incubation with LPS or live Borrelia. PBMC stimulated by LPS increase the net production (stimulated minus unstimulated) of IL-1beta and TNF-alpha significantly, while production of IL-6 was smaller. A delayed increase in the production of IL-10 was also observed. Productions of IL-12 and INF-gamma were not influenced. In contrast to LPS, stimulation of PBMC with live Borrelia, increases also the production of IL-12 and IFN-gamma, besides IL-1beta, TNF-alpha, IL-6 and IL-10. Productions of IL-1beta, IL-6 and TNFalpha increased immediately after incubation with both LPS and Borrelia, while productions of IL-12 and INF-gamma begin to increase 8 hours and production of IL-10 12 hours after stimulation. Data indicate that stimulation with different pyrogens may activate the cells of the immune cascade in a different way. Stimulation of BPMC by LPS seems to activate the initial steps of the immune response (macrophages and granulocytes) only, while infection with live Borrelia also stimulates the later phase of the immune response, probably due to effect of initially produced cytokines.     

Piperine inhibits cytokine production by human peripheral blood mononuclear cells

Piperine, an amide isolated from Piper species (Piperaceae), has been reported to exhibit central nervous system depression, anti-pyretic and anti-inflammatory activity. Immunomodulatory and anti-tumor activity of piperine has been demonstrated in mouse carcinomas. However, there is little information available concerning the effect of piperine on humans. We evaluated the immunopharmacological activity of this compound in human immune cells. Human peripheral blood mononuclear cells (PBMCs) were exposed to piperine, and cell proliferation was determined by the MTS assay. Piperine significantly inhibited phytohemagglutinin-stimulated human PBMC proliferation after exposure for 72 h. This compound inhibited PBMC activity, with an IC(50) of 100.73 ± 11.16 μg/mL. Production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) was measured using an ELISA assay and RT-PCR. Piperine inhibited IL-2 and IFN-γ production in the PBMCs. RT-PCR data indicated that IL-2 and IFN-γ mRNA expression in PBMCs is suppressed by piperine. This compound significantly inhibited the production of these two cytokines by activated PBMCs in a dose-dependent manner. In conclusion, piperine appears to have potential as an immunomodulatory agent for immune system suppression.     

Taurine chloramine modulates cytokine production by human peripheral blood mononuclear cells

Summary.  The effect of taurine (Tau) and taurine chloramine (Tau-Cl) on the production of TNF-α, IL-1β, and IL-6 by peripheral blood mononuclear cells of healthy volunteers was examined. Cells were stimulated with bacterial lipopolysaccharide (LPS) in the presence of either Tau or Tau-Cl. After 24 h culture the cytokine concentrations were measured in both culture supernatants (secreted) and cell lysates (cell-associated) using ELISA. In LPS-stimulated cells Tau-Cl inhibited both the secreted and cell-associated IL-1β and IL-6, while exerted dual effect on TNF-α production: raising it slightly at low and reducing at higher concentration. By contrast, Tau had no significant effect on the cytokine production. These results indicate that Tau-Cl modulates synthesis of pro-inflammatory cytokines, and therefore it may play a role in the initiation and propagation of immune response. Received November 29, 2001 Accepted January 18, 2002 Published online August 30, 2002 Acknowledgments This research was supported by grants from the State Committee for Scientific Research of Poland (No 4 P05B 01018) and the Institute of Rheumatology (No I/14). The Institute of Rheumatology is supported by a core grant from the State Committee for Scientific Research of Poland. Authors' address: Ewa Kontny, Ph.D., Department of Pathophysiology and Immunology, Institute of Rheumatology, Spartanska 1, 02-637 Warsaw, Poland, E-mail: zpatiir@warman.com.pl Abbreviations: Tau, taurine; Tau-Cl, taurine chloramine; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL-1β, interleukin 1β; IL-6, interleukin 6; PBMC, peripheral blood mononuclear cells     

IgE-dependent cytokine production by human peripheral blood mononuclear phagocytes.

These studies demonstrate the IgE-dependent production of IL-1 beta and TNF-alpha by circulating blood monocytes. IL-1 beta production was demonstrated biologically as the stimulation of proliferation of the cloned IL-1-dependent murine T cell line D10.G4.1 in the presence of a submitogenic concentration of PHA. In a representative experiment, 3H-thymidine uptake increased from 57826 cpm in the presence of supernatants obtained from unstimulated cells to 200774 cpm with supernatants from monocytes stimulated by IgE/alpha IgE immune complexes. By ELISA, IgE complexes increased IL-1 beta production from 0.54 +/- 0.06 ng (per 10(6) monocytes) to 2.60 +/- 0.62 ng (p less than 0.01; mean of eight experiments) and TNF-alpha production from 0.17 +/- 0.10 ng to 3.00 +/- 0.54 ng (p less than 0.01; mean of four experiments). No IL-1 alpha secretion was observed. RNA hybridization analysis demonstrated that IL-1 beta production represented de novo synthesis of the cytokine. Stimulated RNA production was observed after a minimal 1/2-h incubation and was maximal at 2 h. The IgE-dependent secretion of these pro-inflammatory cytokines by mononuclear phagocytic cells may contribute to the inflammation characteristic of allergic responses.     

Different modulation by live or killed Helicobacter pylori on cytokine production from peripheral blood mononuclear cells

The mechanisms by which H. pylori colonizes and persists within the gastric mucosa are poorly understood. The induction and maintenance of gastric inflammation appear to depend on the complex interaction between a number of cytokines and chemokines. The gastric immune response observed "in vivo", during H. pylori infection, is characterized by a polarization of Th1 cell type that seems to be responsible for gastric pathology. The purpose of this study was to test the direct effect of H. pylori (live or gentamicin-killed) on human PBMC in order to evaluate the "in vitro" Th1-Th2 balance by monitoring IL-18, IFNgamma and IL-10 production. This study demonstrates for the first time that "in vitro" pretreatment with gentamicin-killed H. pylori of PBMC, followed by infection with live bacteria, downregulates the production of inflammatory cytokines such as IL-18 and IFNgamma Our results provide a possible strategy to restore the immunological disorders determined by H. pylori infection.     

Hypoxia affects cytokine production and proliferative responses by human peripheral mononuclear cells

We have shown that hypoxia (2% O₂ ≈ pO₂ 14 mmHg) as opposed to O₂ atmospheric pressure (20.9% O₂ ≈ pO₂ 140 mmHg) can deeply affect the production of cytokines in human peripheral mononuclear cells (PBMC) in the presence or absence of a specific T-cell activator such as phytohemagglutinin (PHA). In hypoxia, interleukin (IL)-2, IL-4, and interferon (IFN)-γ production increased by 110, 70, and 50% over that of controls, respectively, in PHA-stimulated PBMC (P < 0.05). Moreover, in hypoxia, IL-6 production was significantly enhanced in both resting and PHA-stimulated PBMC by 36 and 37%, respectively (P < 0.05). However, in hypoxia, IL-10 production decreased in both resting and stimulated PBMC, being 80 and 67% of controls, respectively (P < 0.05). PBMC proliferation was not significantly affected by hypoxia, although PBMC susceptibility to PHA was about 80% of that of the control (P < 0.05) after 40 hr of treatment, whereas the cycle progression of hypoxic PBMC was delayed. From an evaluation of these results, hypoxia apparently modifies the production of cytokines by PBMC. These results have both theoretical and practical interest because local hypoxia is very common in several conditions, such as inflammation and local ischemia, and is a host-nonspecific defense against infection. Furthermore, these results suggest a differential pattern of cytokine production in vivo in hypoxic tissues. J. Cell. Physiol. 173:335–342, 1997. © 1997 Wiley-Liss, Inc.     

Enhanced activity of human IL-10 after nitration in reducing human IL-1 production by stimulated peripheral blood mononuclear cells

Nitric oxide and superoxide form the unstable compound, peroxynitrite, which can nitrate proteins and compromise function of proinflammatory cytokines at sites of inflammation. Reduced function of proinflammatory proteins such as IL-8, macrophage inflammatory protein-1alpha, and eotaxin suggest an anti-inflammatory effect of nitration. The effects of nitration on anti-inflammatory cytokines such as IL-10 are unknown. We hypothesized that peroxynitrite would modify the function of anti-inflammatory cytokines like IL-10. To test this hypothesis, the capacity of recombinant human IL-10 to inhibit production of human IL-1beta (IL-1) from LPS-stimulated human PBMC was evaluated. Human IL-10 was nitrated by incubation with peroxynitrite or by incubation with 3-morpholinosydnonimine, a peroxynitrite generator, for 2 h and then incubated with LPS-stimulated PBMC for 6 h, and IL-1 was measured in the culture supernatant fluids. Human IL-1 production was significantly lower in the peroxynitrite- or 3-morpholinosydnonimine-nitrated IL-10 group than in the IL-10 controls (p < 0.05, all comparisons). This finding demonstrates that although peroxynitrite inhibits proinflammatory cytokines, it may augment anti-inflammatory cytokines and further point to an important role for peroxynitrite in the regulation of inflammation.     

Substance P enhances interferon-gamma production by human peripheral blood mononuclear cells

Substance P (SP), a neuropeptide widely distributed in the organism, has been shown to stimulate lymphocyte proliferation and immunoglobulin synthesis. However, the effect of SP on specific lymphokines is unknown. Therefore we investigated the influence of SP on mitogen-induced interferon-gamma (IFN-gamma) production in vitro. Peripheral blood mononuclear cells (PBMC) of healthy donors were isolated by density gradient centrifugation and cultured in supplemented RPMI 1640 medium with phytohemagglutinin (PHA) or pokeweed mitogen (PWM), 0.125 and 0.25 mg/liter each, and varying concentrations of SP (10(-12) to 10(-6) M). After 24 and 48 h, IFN-gamma was measured in the supernatant using radioimmunoassay. Results were expressed as percent change of controls. SP alone had no relevant IFN-gamma inducing properties. It enhanced the IFN-gamma production of PWM-stimulated cells significantly up to 18%. The maximal effect was observed at 10(-8) M. PHA-stimulated cells also increased their IFN-gamma production after addition of SP. However, due to great interindividual variations this effect did not attain statistical significance. Stimulation of IFN-gamma production by SP might be of physiological importance, since the effect was seen at concentrations comparable to those found in the body. Our data lend further support to the immunoregulatory functions of SP.     

Spontaneous inflammatory cytokine gene expression in normal human peripheral blood mononuclear cells

Cytokines regulate cellular immune activity and are produced by a variety of cells, especially lymphocytes, monocytes, and macrophages. Measurement of cytokine levels has yielded useful information on the pathological process of different diseases such as AIDS, endotoxic shock, sepsis, asthma, and cancer. It may also be of use in the monitoring of disease progression and/or inflammation. To determine spontaneous cytokine gene expression in whole blood and PBMCs, whole blood was obtained from healthy volunteers and total mRNA was isolated from PBMCs. The kinetics of response were determined by sequential testing of cytokine gene expression by RT-PCR analysis. Our results demonstrated that isolated and incubated PBMCs expressed TNF-alpha and high levels of IL-1beta, IL-6, IL-8, and IL-10. In contrast, WB only expressed the mRNA cytokines of TNF-alpha and IL-8 (p < 0.05). These results suggest that spontaneous myriad mRNA cytokine expression can be avoided with the use of WB incubation and the rapid collection of PBMCs. Furthermore, this method should be employed in all cases where the levels of cytokine gene expression can be evaluated.     

Anti-peptide antibody production elicited by in vitro immunization of human peripheral blood mononuclear cells

Human monoclonal antibodies have great potential for use in the treatment of various diseases. We have established an in vitro immunization protocol for inducing antigen-specific antibody production from human peripheral blood mononuclear cells (PBMCs). In the in vitro immunization protocol, PBMCs are pretreated with L-leucyl-L-leucine methyl ester (LLME) to remove suppressive cells, and are sensitized and cultured with a soluble antigen in the presence of IL-2, IL-4 and muramyl dipeptide for 8 d, and then an antigen-specific antibody is produced. In this study, we examined the novel possibility of an in vitro immunization protocol, specifically, whether LLME-treated PBMCs can be sensitized with a peptide antigen to produce an anti-peptide antibody. The results indicate that antigen-specific immune responses were elicited by a peptide antigen derived from rice allergen, a cholera toxin B subunit, and TNF-alpha as a sensitizing antigen in in vitro immunization. These results suggest that the in vitro immunization protocol is applicable in the generation of an anti-peptide antibody against various antigens, including food allergens, foreign antigens, and self-antigens.     

Cytokine production by peripheral blood mononuclear cells in recurrent miscarriage

It has been postulated that a proportion of recurrent miscarriage (RM) might be due to immune causes. The objective was to determine whether cytokine expression in peripheral blood mononuclear cell is altered in patients with a history of RM. We compared the levels of IL-2, IL-4, IL-10, IL-13, TGFbeta1 and IFNgamma in the supernatant of Phytohemagglutinin stimulated mononuclear cells in 21 women with RM at the time of 3rd or higher abortion (group I), 32 women who were at least 3 months past their 3rd or higher abortion (group II) and 32 pregnant women with no history of abortion (group III). Gestational age was matched between groups I and III. Group I had higher level of IL-2 than group III (P=0.001). Group II showed higher level of IL-2 (P=0.001) and IFNgamma (P=0.015) than group III. The production of IL-10 by mononuclear cells of group III was higher than both group I (P=0.002) and group II (P=0.001). There was no difference in the levels of IL-2, IL-10 and IFNgamma between groups I and II. Also, the levels of IL-4, IL-13, and TGFbeta1 were similar among the groups. The data indicate an elevation of Th1 cytokines in women with RM as compared to normal pregnant women, and IL-10 is an important cytokine in the maintenance of pregnancy.     

Human peripheral blood mononuclear cells stimulated by Schistosoma mansoni antigens: association between protein tyrosine kinases, mitogen-activated protein kinases and cytokine production

Concerning schistosomiasis, little is known about the intracellular signaling response of human peripheral blood mononuclear cells (PBMC) to Schistosoma mansoni antigens. To understand the critical role of protein tyrosine kinases (PTKs) in PBMC activation by S. mansoni antigens, we investigated how inhibition of PTKs by genistein, a tyrosine kinase inhibitor, affects proliferation, cytokine production and activation of mitogen-activated protein kinases (MAPKs). Our studies showed that PTKs have an important role in proliferation of PBMC from chronic schistosomiasis patients as cells stimulated with S. mansoni soluble antigens in the presence of genistein had an impaired proliferation. In contrast, PTK inhibition failed to cause any effect on MAPKs activity. We also evaluated the cytokine production for interleukin (IL)-2, interferon gamma (IFN-gamma), and IL-10 in culture supernatants of PBMC treated with or without PTKs inhibitor. Our results show that PBMC from chronic patients produced a high amount of IL-10 when stimulated with soluble egg antigen preparation (SEA), however, the amount produced of IL-2 and IFN-gamma was not significant. In the presence of PTKs inhibitor only the production of IL-10 was decreased. The findings suggest that PTKs are involved on signal transduction pathway for PBMC activation, but may not be an absolute requirement for all signaling responses to S. mansoni antigens.     

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.     

Diapocynin versus apocynin as pretranscriptional inhibitors of NADPH oxidase and cytokine production by peripheral blood mononuclear cells

Apocynin has been extensively used as an inhibitor of NADPH oxidase (NOX) in many experimental models using phagocytic and non-phagocytic cells. Currently, there is some controversy about the efficacy of apocynin in non-phagocytic cells, but in phagocytes the reported results are consistent, which could be due to the presence of myeloperoxidase in these cells. This enzyme has been proposed as responsible for activating apocynin by generating its dimer, diapocynin, which is supposed to be the active compound that prevents NADPH oxidase complex assembly and activation.Here, we synthesized diapocynin and studied its effect on inhibition of gp91^phox RNA expression. We found that diapocynin strongly inhibited the expression of gp91^phoxmRNA in peripheral blood mononuclear cells (PBMC). Only at a higher concentration, apocynin was able to exert the same effect. We also compared the apocynin and diapocynin efficacy as inhibitors of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) production in response to lipopolysaccharide (LPS)-activated PBMC. Although apocynin did inhibit TNF-α production, diapocynin had a much more pronounced effect, on both TNF-α and IL-10 production. In conclusion, these findings suggest that the bioconversion of apocynin to diapocynin is an important issue not limited to enzymatic activity inhibition, but also for other biological effects as gp91^phox mRNA expression and cytokine production. Hence, as diapocynin can be easily prepared from apocynin, a one-step synthesis, we recommend its use in studies where the biological effects of apocynin are searched.     

Release of cytokines by human nasal epithelial cells and peripheral blood mononuclear cells infected with Mycoplasma pneumoniae

Mycoplasma pneumoniae (Mp) infection is associated with asthma exacerbation in children. We hypothesized that Mp infection may cause airway inflammation by inducing the release of cytokines by respiratory epithelial cells. The levels of chemokines interleukin-8 (IL-8) and released upon activation, normal t cell expressed and secreted (RANTES) released by nasal epithelial cell (NEC) cultures established from asthmatic and nonasthmatic children were measured by ELISA at 4, 24, 48, and 72 hr after cells were inoculated with Mp, and were compared with baseline release of these factors. The presence of MP on apical membranes of NEC after infection was confirmed by transmission electron microscopy, and adherence was shown to be inhibited by erythromycin. Mp infection did not alter NEC release of IL-8 or RANTES at any time point. In contrast, tumor necrosis factor alpha (TNF-alpha) stimulated increased IL-8 at all time points, and respiratory syncytial virus (RSV) infection stimulated RANTES release at 48 and 72 hr by NEC. These results were not significantly different between NEC from asthmatic and nonasthmatic children. As a comparison, peripheral blood mononuclear cells from normal human volunteers were also incubated with Mp and had significantly increased release of IL-2, IL-6, and TNF-alpha. We conclude that Mp, unlike viral pathogens such as RSV, is unlikely to directly stimulate early airway surface cytokine responses via mechanisms involving epithelial cells. We speculate that the chronic presence of mononuclear cells at the airway surface of asthmatics provides a target for Mp-triggered cytokine production.     

The interactions of phthalocyanines with stimulated and resting human peripheral blood mononuclear cells.

The interactions of two metal-free phthalocyanines [(H2Pc) and Solar Pc (with four peripherical groups: SO2N(CH2CH2OH)2)] and of one metal substituted dye (CoPc) with resting and stimulated human peripheral blood mononuclear cells (PBMC) were compared. The absorption, fluorescence, photoacoustic and EPR spectra of both resting cells and cells stimulated by phytohaemagglutinin, incubated in dimethyl sulfoxide (DMSO) with very low or 95% water content and with or without dye addition, were measured. The fate of the light absorbed by the samples was investigated. It is known that singlet oxygen production is crucial for photodynamic action of dyes. Thermal deactivation and luminescence emission compete with this process, so investigation of these alternative paths of sensitizer deactivation provides information about photodynamic action. The incorporation of the investigated dyes into cells and the perturbation of the cell structure caused by the dyes, the incubation solvent and the activator were investigated by comparing the spectral properties of PBMC before and after stimulation and incubation. Incubation of the cells for 1 h in a solution of Solar Pc in 99.5% aqueous DMSO, resulted in an efficient dye incorporation which was highly selective. Solar Pc being introduced much more efficiently into stimulated cells than into resting cells.     

Anti-angiogenic effects of lycopene through immunomodualtion of cytokine secretion in human peripheral blood mononuclear cells

The carotenoid lycopene has been reported to possess anti-metastatic activity which may be associated with immunomodulation. However, the anti-angiogenic effects and mechanisms of action of lycopene have not been reported. In this study, we investigated the immunomodulatory effect on in vitro and ex vivo angiogenesis of lycopene. We found that the proliferation, migration and the matrigel tube formation of human umbilical vein endothelial cells (HUVECs) was remarkably inhibited by conditioned medium (CM) of human peripheral blood mononuclear cells (MNC-CM) stimulated with various dose (1-10 μmol/L) of lycopene (LP-MNC-CM). LP-MNC-CM treatment inhibited ex vivo angiogenesis, as revealed by chicken egg chorioallantoic membrane assay. We further examined the effects of lycopene stimulation on cytokine levels in MNC and showed that, as compared to the control, lycopene (10 μmol/L) significantly (P<.001) up-regulated interleukin (IL)-12 by 163% and interferon (IFN)-γ by 531%. Furthermore, pre-treatment of HUVECs with dexamethasone, an IL-12 inhibitor, blocked the anti-angiogenic effects of LP-MNC-CM in parallel with inhibition of IL-12 and IFN-γ induction in MNC. These results demonstrate that lycopene has a potent anti-angiogenic effect and that these effect may be associated with its up-regulation of IL-12 and IFN-γ.     

Coculture of human peripheral blood mononuclear cells with Trypanosoma cruzi leads to proliferation of lymphocytes and cytokine production.

Trypanosoma cruzi, which causes Chagas' disease, has been shown to cause polyclonal proliferation of lymphocytes after infection in vivo. This paper demonstrates that coculture of human PBMC with T. cruzi CL strain leads to proliferation of lymphocytes, which peaks on days 5 to 7 after infection. Approximately 15% of lymphocytes in culture undergo blast transformation. The proliferation of lymphoblasts can be measured by [3H]TdR incorporation, because the parasites incorporate little TdR. Parasites derived from autologous PBMC cultures or xenogeneic rat fibroblasts stimulate lymphocyte transformation similarly. By immunofluorescent cytometry, lymphoblasts from these cultures are 23 to 46% B cells (CD19+) and 39 to 64% T cells (CD3+), and approximately half of the T cells are CD4+ and half CD8+. A high percentage of lymphoblasts express MHC class II and IL-2R p55, suggesting both B and T lymphoblasts express these molecules. Anti-MHC class II and anti-IL-2R p55 mAb significantly inhibit the proliferative response of PBMC to T. cruzi. The mRNA for cytokines IL-1 beta, IL-2, IL-5, IL-6, IFN-gamma, and TNF-alpha are detected after T. cruzi coculture with PBMC, peaking on day 3. No IL-4 or IL-10 mRNA are detected. Large quantities of bioactive IL-1 and IL-6 are found in the supernatants of these PBMC. Monocytes, infected in the apparent absence of lymphocytes, assume activated morphology and accumulate mRNA for IL-1 beta, TNF-alpha, and IL-6. T cells require accessory cells to proliferate and produce cytokine mRNA. A trypsin-sensitive activity in lysates of T. cruzi stimulates lymphocyte proliferation. The data presented demonstrate that T. cruzi coculture with PBMC leads to lymphocyte proliferation, monocyte activation, and cytokine production.