Soluble antigens from group B streptococci induce cytokine production in human blood cultures.

Group B streptococcal antigens stimulated tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6 production in human blood cultures in a concentration- and time-dependent fashion. The minimal concentrations of type-specific polysaccharides, lipoteichoic acid, and group-specific polysaccharide required to produce these effects were, respectively, 0.01, 1, and 10 microg/ml. Cell separation experiments indicated that monocytes were the cell type mainly responsible for cytokine production. Time course studies indicated that TNF-alpha was released before the other cytokines. TNF-alpha, however, did not appear to directly induce IL-1beta, as shown by blockade experiments with anti-TNF-alpha antibodies. IL-6 levels were moderately but significantly decreased by anti-TNF-alpha. These data indicate that several products from group B streptococci are able to directly stimulate human monocytes to release TNF-alpha, IL-1beta, and IL-6. These findings may be clinically relevant, since proinflammatory cytokines can mediate pathophysiologic changes during sepsis.     

Extracellular neuraminidase production by group B streptococci.

Neuraminidase (sialidase) activity in concentrated culture filtrates of group B streptococci was measured with bovine submaxillary mucin as substrate. Group B streptococcal neuraminidase was not active on human alpha-1 acid glycoprotein and did not show increased activity on bovine submaxillary mucin that had been O-deacetylated by alkaline treatment. The enzyme was produced in a variety of media, including a chemically defined medium (FMC; Terleckyj et al., Infect. Immun. 11:649-655, 1975) supplemented with bovine serum albumin or human serum albumin. Maximal levels of activity were present in filtrates from cells grown in a dialyzable fraction of Todd-Hewitt broth harvested during the late exponential phase of growth. Dramatic decreases were seen when filtrates from the late stationary phase were assayed. The decrease in specific activity during the stationary phase was shown to be due to proteolytic digestion of neuraminidase and not to the elaboration of an extracellular neuraminic acid aldolase.     

Characterization and incidence of pigment production by human clinical group B streptococci.

Pigment was produced in stab cultures by 97% of 297 group B streptococci isolated from human clinical specimens. The pigment, which was associated with a membranous cell fraction, showed a four-banded absorption spectrum similar to that of a carotenoid, with maxima at 435, 566, 485, and 525 nm. Addition of glucose to the growth medium suppressed pigment production in most strains. Only 37% of strains from bovine sources produced pigment.     

Adherence to, invasion by, and cytokine production in response to serotype VIII group B Streptococci

The adherence to and invasion of the human epithelial cell line A549 by group B streptococcus (GBS) serotype VIII strains were compared with those of serotype III strains by a conventional method and the dynamic in vitro attachment and invasion system. Twenty GBS strains, including nine vaginal isolates and one invasive isolate each of serotypes III and VIII, were used in the conventional attachment and invasion assay. Adherence to and invasion of A549 cells by serotype VIII GBS strains were significantly greater (P < 0.0001) than those by serotype III strains for both the invasive strain and vaginal isolates. Cytokine production by A549 cells following stimulation with GBS serotypes III and VIII or their purified capsular polysaccharides (CPS) was measured. Serotype III strains stimulated significantly greater tumor necrosis factor alpha (TNF-alpha) (P < 0.0001) and interleukin-10 (IL-10) (P < 0.05) production than did serotype VIII strains. IL-8 production in response to serotype VIII was significantly higher (P < 0.001) than that in response to serotype III. TNF-alpha, IL-8, and IL-10 production was greater in A549 cells infected with GBS than in the untreated control cells. TNF-alpha production was significantly greater (P < 0.005) after stimulation with purified GBS serotype III CPS than after stimulation with serotype VIII CPS, a result similar to that after stimulation with whole GBS. IL-12 production by A549 cells was observed only in response to infection with GBS serotype III, resulting in the possibility of a greater TH1 response in serotype III GBS. These results suggest differences in immune responses to infection with GBS serotypes III and VIII.     

Potentiation by human serum of anti-inflammatory cytokine production by human macrophages in response to apoptotic cells

Phagocytosis of apoptotic cells by macrophages leads to the production of anti-inflammatory cytokines, thereby preventing inflammation. In this study, we demonstrate that human serum potentiates the production of anti-inflammatory cytokines, IL-10 and TGF-beta, by PMA-treated THP-1 cells and human monocyte-derived macrophages in response to apoptotic cells, which results in great suppression of the production of proinflammatory cytokine IL-8. Human IgG but not its F(ab)'(2) suppressed the IL-8 production. Pretreatment of macrophages but not apoptotic cells with human serum or human IgG caused the suppression, suggesting that immune complex may not be formed with apoptotic cells. When FcgammaRI was specifically down-modulated by a monoclonal antibody, M22, the potentiating effects of human serum and human IgG on the anti-inflammatory cytokine production and the suppressive effects on IL-8 production were completely abolished. Thus, human IgG and FcgammaRI appear to be critical in leading to the production of anti-inflammatory cytokines by macrophage in response to apoptotic cells.     

Hormonal and type-dependent adhesion of group B streptococci to human vaginal cells.

Adhesion of group B streptococci to epithelial cells of the human vagina proved to be type dependent and to fluctuate during the menstrual cycle with a maximum near the time of ovulation. Oral contraception completely abolished the observed cyclic changes. Reduced serum levels of luteinizing hormones (less than 5 mIU/ml) and of follicle-stimulating hormone (less than 10 mIU/ml) were associated with a 10-fold reduction in adhesion of B streptococci to vaginal cells.     

Bacterial genetics and human immunity to group B streptococci

Serotype III group B Streptococcus agalactiae (GBS) are the most common cause of neonatal sepsis and meningitis. We have classified type III GBS by restriction digest patterns of chromosomal DNA and demonstrated that a subgroup of genetically related strains (RDP type III-3) causes the majority of type III GBS neonatal infection. Genetic differences between type III GBS strains contribute significantly to differences in virulence and host immune responses. While 100% of less virulent RDP type III-1 and III-2 organisms express C5a-ase, an inhibitor of neutrophil chemotaxis, only 63% of virulent RDP type III-3 isolates have functional C5a-ase. Functional differences in type III GBS C5a-ase are attributable to a shared genetic polymorphism, supporting our genetic classification. The mean capsular sialic acid content of virulent RDP type III-3 strains is significantly higher than that of less virulent strains, suggesting that capsular sialylation is also genetically regulated. C5a-ase is not critical for all RDP type III-3 strains to be invasive because the higher capsular sialic acid content of III-3 strains limits complement activation. The identification of these and additional genetic differences between GBS strains has important implications for our understanding of the pathogenesis of these important human infections.     

In vitro cytokine production by mononuclear cells exposed to bone cement extracts

The authors evaluated the ability of bone cement to modify the profile of pro-inflammatory cytokines secreted by the immune cells. Peripheral blood mononuclear cells (PBMC) collected from healthy individuals were cultured with cement extracts and tested to assess the release of IL-1beta, TNFalpha, GM-CSF and IL-6 in both unstimulated and PHA-stimulated PBMC. The cytokine release of unstimulated PBMC was very poor, and in particular the IL-1beta was undetectable: the addition of cement extract increased both TNFalpha and GM-CSF release and decreased IL-6, sometimes significantly. The most recurrent observation in PHA-stimulated PBMCs exposed to bone cement extract was the increase in both IL-1beta and IL-6 release, while both the mean concentration and the index of release of TNFalpha and GM-CSF were changeable. In conclusion our results showed that leachable components of some bone cements can induce in vitro the release of pro-inflammatory cytokines which are known to be involved in the bone resorption associated with aseptic loosening of hip prostheses. These findings allowed us to identify materials endowed with the highest inflammatory power.     

In vitro effect of statins on cytokine production and mitogen response of human peripheral blood mononuclear cells

The in vitro effect of the hydrophilic statin - pravastatin - and three hydrophobic statins - atorvastatin, lovastatin and simvastatin - on the production of IL-1beta, IL-1ra, IL-2, IL-6 and IFN-gamma by human peripheral blood mononuclear cells (PBMC) and their response to mitogens was examined. Lovastatin and simvastatin increased the production of IL-1beta in a dose dependent manner and reduced secretion of IL-1ra at high concentration. These two statins exerted a dose dependent inhibitory effect on IL-2 production and reduced the secretion of IFNgamma at high dose. Atorvastatin did not affect IL-1beta, but suppressed IL-1ra, IL-2 and IFNgamma production. Atorvastatin, lovastatin and simvastatin caused a dose dependent inhibition of mitogen-induced proliferation of PBMC. IL-6 production was not affected by any one of the statins. While pravastatin caused a slight reduction in the proliferative response of PBMC to PHA, it did not affect either their response to Con A and PWM, or the secretion of cytokines tested.     

In vitro effect of lycopene on cytokine production by human peripheral blood mononuclear cells

There is evidence indicating that regular consumption of tomato products is associated with favorable immunomodulatory effects. In addition, tomato extracts have been shown to possess antioxidant, anticarcinogenic and antithrombotic activity in vitro. Since tomatoes are rich in carotenoids and particularly in lycopene--the pigment responsible for the red color of tomatoes--the present work was designed to examine the in vitro effect of lycopene on cytokine production by peripheral blood mononuclear cells (PBMC) from 15 healthy subjects. First, 2 x 10(6) PBMC suspended in 1 ml of conditioned medium were incubated over a period of 24 and 48 hours without or with the following concentrations of lycopene: 0.25, 0.5, 1.0, 2.0 and 4.0 microM. The production of the subsequent cytokines was evaluated: IL-1beta, IL-1ra, IL-2, IL-6 and IL-10, as well as TNFalpha and IFNgamma. Lycopene induced a dose-dependent increase in IL1beta, and TNFalpha production and a decrease in IL-2, IL-10 and IFNgamma secretion, whereas that of IL-6 and IL-1ra was not affected. It is concluded that understanding the role of lycopene in modulation of the immune system may promote decisions as for dietary supplementation of lycopene for reducing the risk of certain diseases.     

Impaired T cell activation and cytokine production by calcitriol-primed human B cells

The biologically active form of vitamin D₃, 1, 25-dihydroxyvitamin D₃ (calcitriol), is a potent modulator of the immune response. We have shown previously that calcitriol modulates the immunoglobulin response in vitro and in vivo in mice and humans. To analyse the underlying molecular mechanisms we studied whether calcitriol-primed B cells modulate T cell activation and function. Human B cells were stimulated with anti-CD40 and interleukin (IL)-4 in the presence of increasing concentrations of calcitriol. After removal of calcitriol, primed B cells were co-cultured with autologous CD4⁺ T cells; the B cell phenotype T cell activation and their consecutive cytokine production were also assessed. Naive T cells co-cultured with calcitriol-primed naive B cells showed a reduced expansion, nuclear factor of activated T cells, cytoplasmic 2 (NFATc2) expression and cytokine production upon restimulation. CD86 expression on B cells after calcitriol priming was identified as an underlying mechanism, as T cell activation and expansion was rescued by activating anti-CD28 antibodies. Our data indicate that calcitriol-primed B cells display an impaired capacity to activate T cells. Taken together, we identified a novel B cell-dependent vitamin D immune regulatory mechanism, namely by decreased co-stimulation of calcitriol-primed B cells.     

Factors influencing adherence of group B streptococci to human vaginal epithelial cells.

Factors affecting the adherence of group B streptococci to human vaginal epithelial cells in vitro were examined. Maximal adherence was achieved within 15 min of incubation of bacteria with epithelial cells. Adherence was temperature and pH dependent; maximal adherence occurred at 37 degrees C and pH 5.5. Killing of streptococci with ultraviolet light or penicillin did not affect adherence. Similarly, adherence was not altered by preincubating epithelial cells at 65 degrees C for 30 min. Thus neither bacterial nor epithelial cell viability appears to be a prerequisite for adherence. Preincubation of streptococci at 65 degrees C for 30 min resulted in a marked decrease in adherence, whereas preincubation of group B streptococci with neuraminidase was associated with a significant increase in adherence. The adherence of strains belonging to five different group B streptococcal serotypes was not altered by group-specific or type-specific rabbit antisera. These findings suggest that the site for adherence on the bacterial cell wall is heat sensitive and is marked by sialic acid, but is not related to either group-specific or type-specific antigens.     

Induction of cyclooxygenase-2 by human monocytes exposed to group B streptococci

Group B streptococcal (GBS) infections are associated with high morbidity and mortality. The molecular pathways mediating the pathophysiological events in GBS infection are not fully delineated. Cyclooxygenases (COX) are the enzymes that convert arachidonate to active eicosanoids. To identify the effects of GBS on eicosanoid metabolism and regulatory mechanisms, we exposed human monocytes to GBS and found that they secreted prostaglandin E2, prostacyclin, and thromboxane A2. Exposure to GBS caused monocytes to express COX-2 mRNA and protein in both a time- and concentration-dependent manner that correlated with eicosanoid production. COX-1 protein was unchanged. Addition of the anti-inflammatory cytokines interleukin (IL)-4 or IL-10 markedly attenuated GBS-induced COX-2 protein accumulation after GBS exposure, as did inhibition of p38 MAPK. Our experiments are the first to show that exposure of monocytes to a gram-positive bacterium (GBS) results in induction of functional COX-2, suggesting that eicosanoids may play important roles in the pathogenesis of GBS infections.     

Acellular components of Chlamydia pneumoniae stimulate cytokine production in human blood mononuclear cells

Accumulating evidence suggest that infection with Chlamydia pneumoniae is associated with atherosclerosis, but the mechanisms involved remain unclear. Inflammation is important in the initial phase of atherogenesis, and cytokines are important in the initiation and progression of inflammation. The aim of this study was to assess the capacity of acellular components of C. pneumoniae to stimulate the production of pro-inflammatory cytokines and chemokines. Peripheral blood mononuclear cells were stimulated in vitro with sonicated C. pneumoniae. Significant amounts of TNF-alpha, IL-1, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) were produced. Inhibition of endotoxin using polymyxin B revealed that chlamydial endotoxin plays a minor role in the cytokine induction. Neutralization of TNF by TNF-binding protein and blockade of IL-1 receptors by IL-1 receptor antagonist revealed that TNF, IL-1 and IL-6 production was independent from each other, whereas IL-8 synthesis was strongly dependent on endogenous TNF and IL-1. In contrast, synthesis of MCP-1 and MIP-1alpha was dependent on endogenous TNF, but not IL-1. In conclusion, acellular components of C. pneumoniae are a potent stimulus for cytokine production, and this mechanism may have an important role in the inflammatory aspects of atherogenesis.     

Kinetics of hemolysin production in bovine group B streptococci

Thirteen strains of group B streptococci etiologically related to bovine infections were investigated in order to observe the effects of non-hemoglobin iron and glucose on growth and hemolysin production; and to determine the necessity to stabilize the hemolysin with a carrier-stabilizer agent. Ferric citrate was diluted to give final concentrations of 1-11 micrograms/ml, added to iron-reduced (by CaCl2 precipitation) brain heart infusion media, inoculated and incubated at 37 degrees C. Parallel experiments were carried out with glucose. A variety of agents was employed to act as hemolysin stabilizers. Hemolysin production was detected by lysis of sheep erythrocytes. Both iron and glucose were crucial in concentrations 5-7 micrograms/ml and 0.5-1% respectively. Bovine serum albumin-starch mixture acted as an excellent stabilizer.     

Intracellular survival of persistent group A streptococci in cultured epithelial cells

Group A streptococcus (GAS) is the principle etiologic agent of bacterial pharyngotonsillitis and a wide range of other diseases. Failure to eradicate GAS from patients has been documented in 5-30% of patients with pharyngotonsillitis, in spite of the continued sensitivity of GAS to penicillin and other beta-lactams. It was recently proposed that eradication failure might be attributed to the ability of GAS to maintain an intracellular reservoir during antibiotic treatment. We have previously shown that strains derived from patients with bacterial eradication failure, despite antibiotic treatment (persistent strains), adhered to and were internalized by cultured epithelial cells more efficiently than strains that were successfully eradicated. Since, penicillin and other beta-lactams do not penetrate well into mammalian cells, intracellular survival of GAS is crucial in order to persist during prolonged antibiotic treatment. In this study, we compared the survival of GAS strains from cases of eradication failure and eradication success, using an epithelial cell culture model. We found that persistent strains show significantly increased intracellular survival, compared to the 'eradication success' strains. This finding supports the idea that an intracellular reservoir of GAS plays a role in the etiology of antibiotic eradication failure.     

Effects of activin A on IgE synthesis and cytokine production by human peripheral mononuclear cells.

Activin A not only stimulates the synthesis and release of pituitary follicle-stimulating hormone, but exerts various effects on haematopoietic cells, embryos, and fibroblasts. In the present study we have examined effects of activin A on IgE synthesis and cytokine production by peripheral blood mononuclear cells (PBMC) in normal humans. When PBMC were cultured in the presence of IL-4, activin A significantly augmented IgE production induced by IL-4. Activin A did not affect, however, IgE production from highly purified B cells when they were stimulated with anti-CD40 MoAb and IL-4. The fact that in the latter condition IgE synthesis was T cell- and monocyte-independent indicated that activin A does not directly influence B cells for IgE synthesis. Rather, production as well as gene expression of IL-6, which is known to enhance IgE synthesis by purified monocytes, was induced by activin A alone. In addition, activin A induced other monokines such as IL-1 and tumour necrosis factor (TNF)-alpha from monocytes. In contrast, activin A neither induced nor augmented the production of TNF-beta or interferon-gamma (IFN-gamma), both of which are known to be exclusively generated by T cells. These data indicate that activin A plays a certain role in physiological functions for monocytes in normal humans.     

Cytokine production and expression of leucocyte-differentiation antigens by human mononuclear cells in response to mycobacterium tuberculosis antigens

The aim of this work was to characterize a leucocyte-differentiation antigen or chemokine receptor that allows the identification of type 1 (T helper 1 (Th1), Tc1) and type 2 (Th2, Tc2) lymphocytes in short-term-cultured human peripheral blood mononuclear cells. In addition, we assessed the type of response induced by mycobacterial antigens in tuberculosis patients and healthy contacts. Cells were stimulated with an unfractionated culture filtrate or 30 kDa antigen from Mycobacterium tuberculosis. Then, CD4 and CD8 cell labelling was combined with CD30, CD27, CD28, CD45RA or CD45R0 staining, detection of intracellular interferon-gamma (IFN-gamma) or interleukin-4 (IL-4) and analysis by three-colour flow cytometry. In separate experiments, the expression of different chemokine receptors (CCR1, CCR3, CCR5, CXCR3 and CXCR4) was also studied. We found that none of the cell-surface molecules studied was preferentially expressed by Th1 or Th2 cells. Thus, our results indicate that these lymphocyte subsets cannot be identified in short-term-cultured mononuclear cells on the basis of preferential expression of the cell markers studied, and that it is necessary to look for additional molecules that allow the discrimination of Th1 and Th2 cells.     

Detection and identification of group B streptococci by use of pigment production

Pigment production by group B streptococci (GBS) is a useful test for identification of the organisms. The test is positive in 99.5% of beta-haemolytic strains. No false-positives are noted. Non-haemolytic strains do not produce pigment. Islam's media less agar can be used as a one-step broth detector of GBS in mixed cultures. This may have application for the detection of GBS in women in labour. When used as an identification system for GBS, serum-starch broth can be further modified by reduction of serum and starch concentrations by at least 80%.     

Relationship between pigment production and haemolysin formation by Lancefield group B streptococci

Group B streptococci produce both a pigment and a haemolysin. The requirements of group B streptococci for the formation and release of pigment and haemolysin are similar and have been examined to extend observations on the relationship between the two products. The amount of pigment and haemolysin extractable from actively metabolising washed-cell suspensions of group B streptococci varied with the atmosphere of incubation, the pH at which the extraction was carried out and the presence of Mg2+ ions. Both pigment and haemolysin were produced in significant amounts in all phases of the growth cycle. When conditions were established for obtaining maximum yields of haemolysin, its production correlated closely with pigment yields, but pigment did not function as a carrier for haemolysin. Formation of pigment, but not of haemolysin, increased in the presence of trimethoprim or higher concentrations of glucose. The composition of pigment produced in different conditions differed qualitatively and different strains of group B streptococci formed pigment of different appearance, suggesting that group B streptococcal pigment is composed of several different substances.