Purified capsular polysaccharide of Cryptococcus neoformans induces interleukin-10 secretion by human monocytes.

In this study, we demonstrated that purified capsular polysaccharide of Cryptococcus neoformans is a potent inducer of interleukin-10 (IL-10) secretion by human monocytes. Endogenous IL-10 was involved in regulating tumor necrosis factor alpha and IL-1beta secretion by human monocytes in response to encapsulated C. neoformans strains. Our results suggest a new immunosuppressive effect exerted by glucuronoxylomannan through the induction of IL-10, a potent downregulator of proinflammatory cytokines.     

Effect of xanthine derivates and dexamethasone on Streptococcus pneumoniae-stimulated production of tumor necrosis factor alpha, interleukin-1 beta (IL-1 beta), and IL-10 by human leukocytes.

The present study concerns the release of the proinflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha and of the anti-inflammatory cytokine IL-10 by human leukocytes in whole blood during stimulation with Streptococcus pneumoniae and the effects of various xanthine derivates, i.e., pentoxifylline (PTX), caffeine, and theofylline, and of dexamethasone (DXM). All three xanthine derivates and DXM inhibited the release of tumor necrosis factor alpha, PTX being the most effective. PTX, theofylline, and DXM inhibited the release of IL-1 beta, but caffeine did not affect IL-1 beta release. The release of IL-10 was significantly reduced by PTX at 24 h and by caffeine at 48 h, but DXM increased the release of this cytokine. In sum, the results of this study demonstrate that DXM inhibits only the release of proinflammatory cytokines but not of the anti-inflammatory cytokine IL-10 by human leukocytes, while PTX is the most potent inhibitor of both proinflammatory and anti-inflammatory cytokines.     

Comparative study of cytokine release by human peripheral blood mononuclear cells stimulated with Streptococcus pyogenes superantigenic erythrogenic toxins, heat-killed streptococci, and lipopolysaccharide.

The differences between toxic or septic shocks in humans during infections by streptococci and gram-negative bacteria remain to be fully characterized. For this purpose, a quantitative study of the cytokine-inducing capacity of Streptococcus pyogenes erythrogenic (pyrogenic) exotoxins (ETs) A and C, heat-killed S. pyogenes bacteria, and Neisseria meningitidis endotoxin (lipopolysaccharide [LPS]) on human peripheral blood mononuclear cells (PBMC) and monocytes has been undertaken. The levels of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), and TNF-beta induced by these bacterial products and bacteria were determined by using cell supernatants. The capacity of ETs to elicit the monocyte-derived cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha was found to depend on the presence of T lymphocytes, because of the failure of purified monocytes to produce significant amounts of these cytokines in response to ETs. PMBC elicited large amounts of these cytokines, as well as IL-8 and TNF-beta, with an optimal release after 48 to 96 h. The most abundant cytokine produced in response to ETA was IL-8. In contrast to the superantigens ETA and ETC, LPS and heat-killed streptococci stimulated the production of significant amounts of IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha, with optimal production after 24 to 48 h in monocytes, indicating no significant involvement of T cells in the process. ETs, but neither LPS nor streptococci, were potent inducers of TNF-beta in PBMC. This study outlines the differences in the pathophysiological features of shock evoked by endotoxins and superantigens during infection by gram-negative bacteria and group A streptococci, respectively. The production of TNF-alpha was a common pathway for LPS, streptococcal cells, and ETs, although cell requirements and kinetics of cytokine release were different.     

Induction of proinflammatory cytokines from human respiratory epithelial cells after stimulation by nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) causes repeated respiratory infections in patients with chronic lung diseases. These infections are characterized by a brisk inflammatory response which results in the accumulation of polymorphonucleated cells in the lungs and is dependent on the expression and secretion of proinflammatory cytokines. We hypothesize that multiple NTHi molecules, including lipooligosaccharide (LOS), mediate cellular interactions with respiratory epithelial cells, leading to the production of proinflammatory cytokines. To address this hypothesis, we exposed 9HTEo- human tracheal epithelial cells to NTHi and compared the resulting profiles of cytokine gene expression and secretion using multiprobe RNase protection assays and enzyme-linked immunosorbent assays (ELISA), respectively. Dose-response experiments demonstrated a maximum stimulation of most cytokines tested, using a ratio of 100 NTHi bacterial cells to 1 9HTEo- tracheal epithelial cell. Compared with purified LOS, NTHi bacterial cells stimulated 3.6- and 4.5-fold increases in epithelial cell expression of interleukin-8 (IL-8) and IL-6 genes, respectively. Similar results were seen with epithelial cell macrophage chemotactic protein 1, IL-1alpha, IL-1beta, and tumor necrosis factor alpha expression. Polymyxin B completely inhibited LOS stimulation but only partially reduced NTHi whole cell stimulation. Taken together, these results suggest that multiple bacterial molecules including LOS contribute to the NTHi stimulation of respiratory epithelial cell cytokine production. Moreover, no correlation was seen between NTHi adherence to epithelial cells mediated by hemagglutinating pili, Hia, HMW1, HMW2, and Hap and epithelial cytokine secretion. These data suggest that bacterial molecules beyond previously described NTHi cell surface adhesins and LOS play a role in the induction of proinflammatory cytokines from respiratory epithelial cells.     

Mycoplasma arginini TUH-14 membrane lipoproteins induce production of interleukin-1, interleukin-6, and tumor necrosis factor alpha by human monocytes.

Mycoplasma arginini TUH-14 partially purified membrane lipoproteins (TUH-14-pp) directly induce secretion of the cytokines involved in the inflammatory response, namely, interleukin 1 (IL-1), tumor necrosis factor alpha, and IL-6, by human monocytes cultured in the absence of serum. The biological activity of each cytokine correlates with its immunoreactivity. Upon stimulation with either TUH-14-pp or lipopolysaccharide, most tumor necrosis factor alpha and IL-6 is secreted in the extracellular compartment, whereas a significant amount of IL-1 remains cell associated. Finally, polymyxin B does not affect secretion of cytokines induced by TUH-14-pp, indicating that mycoplasma lipopolysaccharide does not account for their effects on monocytes. Altogether, our data show that direct interaction of mycoplasma membrane components with human blood monocytes induces secretion of high levels of cytokines known to trigger inflammatory responses. This new concept of membrane-bound active components of mycoplasma may explain its ability to efficiently initiate inflammatory reactions.     

Cytokine mRNA expression in peripheral blood cells of immunosuppressed human islet transplant recipients

The macrophage derived cytokines interleukin-1 beta (IL-1β), and tumor necrosis factor alpha (TNFα), and the T-cell derived cytokine interferon gamma (IFNγ) have been implicated to play an important role in early attack on islet cells during human islet transplantation (ITx). Therefore, the aim of this study was to investigate the influence of the current immunosuppressive induction therapy in clinical islet transplantation on mRNA expression of these cytokines in blood cells, compared to lipopolysaccharide (LPS) induced cytokine release in vitro and to plasma levels. The cytokine release correlated to lymphocyte counts and significantly decreased after ATG, and partially recovered 2 weeks after ITx. Unexpectedly, there was no correlation between mRNA expression for IL-1β in total blood and the number of lymphocytes and monocytes remaining after anti thymocyte globulin (ATG)-therapy. Even when the blood was nearly totally depleted from mononuclear cells, high amounts of IL-1β mRNA could be detected. However, IL-1β secretion could not be stimulated in vitro. Our results show that application of ATG during ITx might contribute to graft survival during the early posttransplant period by suppression of the synthesis of monocyte derived cytokines IL-1β and TNFα.     

Selective cytokine production by epithelial cells following exposure to Escherichia coli.

This study compared the repertoire of cytokines produced by epithelial cell lines and human peripheral blood monocytes in response to Escherichia coli. The A-498 and J82 urinary tract epithelial cell lines and human peripheral blood monocytes were exposed to E. coli Hu734. The cytokine content of single cells was detected by indirect immunofluorescence using monoclonal antibodies to interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor alpha (TNF-alpha), TNF-beta, IL-6, IL-8, and granulocyte macrophage-colony-stimulating factor, and the number of positive cells was used to quantitate the response. The J82 bladder cell line stained positive for IL-6, IL-8, and IL-1 alpha. The IL-8 and IL-6 response peaked at 2 h, while the number of IL-1 alpha-positive cells reached a peak 6 h after E. coli stimulation. The A-498 kidney cell line stained for IL-8 with a peak at 2 h and IL-6 with a peak at 6 h after E. coli stimulation. Peripheral blood monocytes stained for the cytokines IL-1 alpha, IL-1 beta, IL-8, IL-6, and TNF-alpha but not for TNF-beta and granulocyte macrophage-colony-stimulating factor after stimulation with E. coli. The results demonstrated that bacteria activated a cytokine response in the epithelial cell lines and monocytes. The epithelial cell lines had a more limited cytokine response profile than circulating monocytes, which may serve to limit the consequences of microbial exposure at the mucosal surface and help maintain the integrity of other tissue compartments.     

Insulin-like growth factor-I stimulates IL-10 production in human T cells

There is vast body of evidence that the insulin-like growth factor (IGF)-I exerts immunomodulatory effects in vitro and in vivo. In vitro studies indicate that stimulatory effects of IGF-I may be exerted through augmentation of inflammatory cytokine production. To further explore the immunomodulatory effects of IGF-I through regulation of cytokine production, we tested the in vitro effects of IGF-I on the secretion of inflammatory T helper cell type 1 (Th1) and Th2 cytokines by human peripheral blood mononuclear cells (PBMC). To this end, PBMC were stimulated with the T cell mitogen phytohemagglutinin (PHA), and cytokines in the culture media were assessed after 18, 42, 66, and 80 h of culture. We found that IGF-I stimulated the secretion of the Th2 cytokine interleukin (IL)-10 by 40-70% in PHA-stimulated PBMC. In addition, we observed a small stimulatory effect (15%) on the secretion of another Th2 cytokine IL-4. The secretion of IL-2, IL-5, IL-6, interferon-gamma, and the inflammatory cytokines IL-1beta, IL-8, and tumor necrosis factor alpha was not or was hardly affected. IL-10 secretion was also stimulated in purified T cells, and we established that IGF-I also stimulated IL-10 mRNA expression by 100-150%. The monocyte-activating bacterial cell-wall product lipopolysaccharide induced IL-10 production in PBMC, but this was not affected by IGF-I. As IL-10 predominantly exerts anti-inflammatory actions and suppresses Th1-dependent immune responses, our results indicate that IGF-I may exert inhibitory actions on inflammatory and Th1-mediated cellular immune responses through stimulation of IL-10 production in T cells.     

Human macrophage response to UHMWPE, TiAlV, CoCr, and alumina particles: analysis of multiple cytokines using protein arrays

Aseptic loosening of total joint replacements is believed to be initiated by a macrophage response to prosthetic wear debris. To better characterize the early response to clinically relevant wear debris, we challenged primary human macrophages from four donors with ultra high molecular weight polyethylene (UHMWPE), TiAlV, CoCr, and alumina particles. After a 24-h culture, protein arrays were used to quantify the secretion of 30 different cytokines and chemokines. Macrophages secreted detectable levels of nine mediators in culture: Interleukin-1alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha), IL-1beta, MCP-1, IL-8, IL-6, GM-CSF, IL-10, and IL-12p40. TiAlV particles were the most stimulatory, causing 5- to 900-fold higher cytokine expression compared with nonstimulated cells and uniquely eliciting high levels of IL-1alpha, IL-6, IL-10, and GM-CSF. CoCr and alumina were mildly stimulatory and typically elicited two- to fivefold greater levels than nonstimulated cells. Surprisingly, UHMWPE did not elicit a significant increase in cytokine release. Our data suggests that IL-1alpha, TNF-alpha, IL-1beta, and MCP-1 are the primary initiators of osteolysis and implicates metallic debris as an important trigger for their release.     

IL-1alpha,IL-1beta and TNF-alpha secretion during in vivo/ex vivo cellular interactions with titanium and copper

Titanium (Ti) and copper (Cu) were used to evaluate cytokine secretion around materials with different chemical properties. Ti disks were coated with Cu or left uncoated. The disks were inserted subcutaneously in rats for 1, 3, 12, 18, 24 and 48 h. Interleukin-1alpha (IL-1alpha), IL-1beta and tumor necrosis factor-alpha (TNF-alpha) concentrations were measured in vivo around the materials, in sham operated sites, and after ex vivo incubation of surface adherent cells. Ti and Cu revealed distinct cytokine expression patterns. Cu recruited cells showed higher and prolonged release of IL-1alpha than Ti at longer times (>24 h), whereas Ti exhibited a transient IL-1alpha response at earlier periods (<24 h). An early enhanced secretion of TNF-alpha characterized Ti. Low amounts of IL-1beta were found around both materials. Sham site recruited cells produced lower levels of cytokines. The results after ex vivo incubations were similar to those in vivo. This study shows that material chemical properties influence early cytokine production. The Ti-associated transient rise of IL-1alpha and TNF-alpha may be of importance for the early tissue response around biocompatible materials, while a delayed high IL-1alpha expression could be a marker of inflammation induced by toxic materials.     

Temporal sequence and kinetics of proinflammatory and anti-inflammatory cytokine secretion induced by toxic shock syndrome toxin 1 in human peripheral blood mononuclear cells.

The staphylococcal superantigen toxic shock syndrome toxin 1 (TSST-1) induces massive cytokine production, which is believed to be the key factor in the pathogenesis of TSS. The temporal sequence and kinetics of both proinflammatory and anti-inflammatory cytokines induced by TSST-1 in human peripheral blood mononuclear cells were investigated. A panel of loss-of-function single-amino-acid-substitution mutants of TSST-1, previously demonstrated to be defective in either major histocompatibility complex (MHC) class II binding (G31R) or T-cell receptor (TCR) interaction (H135A, S14N), was studied in parallel to further elucidate the mechanisms of cytokine secretion. Wild-type recombinant (WT r) TSST-1 induced a biphasic pattern of cytokine secretion: an early phase with rapid release of proinflammatory cytokines (especially gamma interferon, interleukin-2 [IL-2], and tumor necrosis factor alpha [TNF-alpha]) within 3 to 4 h poststimulation, and a later phase with more gradual production of both proinflammatory (IL-1beta, IL-12, and TNF-beta) and anti-inflammatory (IL-6, IL-10) cytokines within 16 to 72 h poststimulation. G31R, which is defective in MHC class II binding, induced a cytokine profile similar to that of WT rTSST-1, except that secretion of the early-phase proinflammatory cytokines was delayed and production of IL-1beta and IL-12 was markedly reduced. In contrast, mutant toxins defective in TCR interaction either demonstrated complete absence of any cytokine secretion during the entire observation period (H135A) or resulted in complete abolishment of IL-2 and other early-phase proinflammatory cytokines, while secretion of IL-10 appeared unaffected (S14N). Neither WT rTSST-1 nor the mutant toxins induced IL-4 or transforming growth factor beta. Our data indicate that effective TCR interaction is critical for the induction of the early-phase proinflammatory cytokine response, thus underscoring the importance of T-cell signaling in TSS.     

Downregulation by cryptococcal polysaccharide of tumor necrosis factor alpha and interleukin-1 beta secretion from human monocytes.

The regulation by Cryptococcus neoformans encapsulation of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) production by human monocytes was investigated. By using encapsulated and acapsular C. neoformans, we demonstrated that both strains induce cytokine production, although the acapsular strain was a better stimulator than the thinly encapsulated strain. The cytokine levels produced by cells stimulated by the two strains were lower and followed a different kinetic than those stimulated by lipopolysaccharide (LPS). Purified capsular polysaccharide inhibits TNF-alpha secretion induced by LPS or acapsular C. neoformans. In contrast, no regulator effect on IL-1 beta was observed when LPS was used. The secretory response of these cytokines follows different pathways of macrophage activation; in fact, complete inhibition of TNF-alpha does not affect IL-1 beta production and vice versa. These data indicate that purified capsular polysaccharide of C. neoformans could contribute to the in vivo progress of cryptococcosis by suppressing cytokine production of macrophages and suggest that a therapeutic approach to address the suppressive effect of cryptococal polysaccharide could be devised.     

Increased concentrations of interleukin-6 and interleukin-1 receptor antagonist and decreased concentrations of beta-2-glycoprotein I in Gambian children with cerebral malaria.

To investigate the pathogenic versus the protective role of cytokines and toxin-binding factors in Plasmodium falciparum infections, we measured the concentrations of tumor necrosis factor alpha, interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-1 receptor antagonist, and IL-6, as well as soluble receptors of tumor necrosis factor and IL-6 (sIL-6R) in serum of Gambian children with cerebral malaria, mild or asymptomatic malaria, or other illnesses unrelated to malaria. Because cytokine secretion may be triggered by toxic structures containing phosphatidylinositol (PI), we also measured concentrations of anti-PI antibodies and the PI-binding serum protein beta-2-glycoprotein I. We found increased concentrations of IL-6, sIL-6R, IL-1ra, and some immunoglobulin M antibodies against PI in children with cerebral malaria, but those who died had decreased concentrations of beta-2-glycoprotein I. We conclude that increased concentrations of cytokines and soluble cytokine receptors represent a normal host response to P. falciparum infections but that excessive secretion of cytokines like IL-6 may predispose to cerebral malaria and a fatal outcome while beta-2-glycoprotein I may protect against a fatal outcome of cerebral malaria.     

Measurement of immunoreactive interleukin-1 beta from human mononuclear cells: optimization of recovery, intrasubject consistency, and comparison with interleukin-1 alpha and tumor necrosis factor

Numerous studies have reported altered levels of in vitro production of the cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF) from blood leukocytes in various human disease states. Most of these studies have used bioassays which are vulnerable to inhibitors produced by these cells. Furthermore in vitro cytokine production is often assessed on a single occasion. The present study was designed to standardize stimulation conditions for in vitro IL-1 beta production and to employ a competitive radioimmunoassay (RIA) to demonstrate reproducibility and long-term variation of in vitro cytokine production in a cohort of healthy human subjects. We also examined relative amounts of immunoreactive IL-1 beta, IL-1 alpha, and TNF induced by the stimuli endotoxin, phytohemagglutinin, or Staphylococcus epidermidis. We show that the RIA can reliably detect IL-1 beta produced from mononuclear cells in concentrations as low as 115 pg/ml. Lysing cells by repeated freeze-thawing yields maximal recovery of total (i.e., secreted plus cell-associated) immunoreactive IL-1 beta, when compared to extraction with the detergent CHAPS or addition of protease inhibitors. Repeated measurement of in vitro cytokine production on different days within 1 week shows good reproducibility for a given individual and a given stimulus (variation coefficient 20 to 30%). Over a long time period (6 months) in vitro cytokine production is stable in some individuals but changes considerably in others. The soluble stimulus endotoxin induces twofold more IL-1 alpha than IL-1 beta or TNF; in contrast the phagocytic stimulus heat-killed S. epidermidis induces fourfold more IL-1 beta and TNF than IL-1 alpha. This distinct pattern of cytokine response indicates differential stimulation of the mononuclear cells by different stimuli. The results form the basis for studying in vitro cytokine production in different human disease states.     

Interleukin-6 production by human monocytes stimulated with Cryptococcus neoformans components.

In order to ascertain if Cryptococcus neoformans components can induce interleukin-6 (IL-6) production, we stimulated human whole blood with purified capsular products. Their potencies in stimulating IL-6 release were mannoproteins > galactoxylomannan = glucuronoxylomannan > alpha(1-3)glucan. IL-6 production was tumor necrosis factor alpha independent and required the presence of monocytes and plasma. Since IL-6 can stimulate replication of the human immunodeficiency virus in monocytic cells, these findings may be clinically relevant.     

Murine macrophages differentially produce proinflammatory cytokines after infection with virulent vs. avirulent Legionella pneumophila

Virulent Legionella pneumophila replicate readily in thioglycollate-elicited peritoneal macrophages from genetically permissive A/J mice, but avirulent L. pneumophila do not. The production of cytokines by macrophages infected with L. pneumophila has been studied, but the correlation of bacterial virulence with immune responses of macrophages, such as proinflammatory cytokine production, is not well understood. In this regard, production of the cytokines tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1alpha, IL-1beta, and IL-6 were examined in macrophage cultures infected in vitro with virulent vs. avirulent L. pneumophila. Infection of macrophages from A/J mice with the virulent L. pneumophila up-regulated mRNA expression for these cytokines, whereas avirulent bacteria resulted in only a slight or no detectable increase in cytokine mRNA. Similarly, virulent L. pneumophila induced the macrophages to produce relatively high levels of TNF-alpha, IL-1alpha, IL-1beta, and IL-6 proteins as measured by enzyme-linked immunosorbent assays, whereas avirulent bacteria induced only low or often undetectable amounts of these cytokines. Thus, these results show the murine macrophages from susceptible A/J mice are readily infected with virulent L. pneumophila in vitro and stimulated to produce the proinflammatory acute-phase cytokines TNF-alpha, IL-1alpha, IL-1beta, and IL-6, but avirulent L. pneumophila did not. Such differences in induction of these proinflammatory cytokines by macrophages in response to virulent vs. avirulent L. pneumophila infections may be an important factor in the pathogenesis induced by these intracellular bacteria.