Possible role of Toll-like receptor-2 in the intracellular survival of Staphylococcus aureus in murine peritoneal macrophages: involvement of cytokines and anti-oxidant enzymes.

Effects of blocking toll-like receptor-2 (TLR-2) on the survival of Staphylococcus aureus (S. aureus) and cytokine production in peritoneal macrophages of Swiss albino mice were analysed. Macrophages were infected with S. aureus in the presence and absence of anti-TLR-2 antibody. Tumour necrosis factor-α (TNF-α) interleukin-6 (IL-6), interferon-gamma (IFN-γ), interleukin-1ß (IL-1ß), interleukin-12 (IL-12) and interleukin-10 (IL-10) concentrations were measured. Expressions of TLR-2, NF-κB, MyD 88 were analysed by Western Blot. Expression of TLR-2 was increased in S. aureus-infected macrophages with respect to control and was MyD 88 independent. TLR2 blocking significantly reduced TNF-α, IL-6, IL-1ß and IL-10 and increased IFN-γ and IL-12 production. Decreased catalase activity and increased superoxide dismutase (SOD) by S. aureus with concomitant increase in H2 O2 and nitric oxide (NO) were observed in the case of prior TLR-2 blocking. To understand whether catalase contributing in the intracellular survival, was of bacterial origin or not, 3-amino, 1, 2, 4-triazole (ATZ) was used to inhibit specifically macrophage-derived catalase. Catalase enzyme activity from the whole staphylococcal cells in the presence of ATZ suggested that the released catalase were of extracellular origin. From the intracellular survival assay, it was evident that pretreatment of macrophages with ATZ reduces the bacterial burden in macrophages when infected with the recovered bacteria only from the anti-TLR-2 antibody-treated macrophages after phagocytosis. Catalase protein expression from the whole staphylococcal cells recovered after phagocytosis also indicated the catalase release from S. aureus. Capturing of S. aureus via TLR-2 induces inflammatory reactions through activation of NF-κB-signalling pathways which was MyD88-independent.

Gentamicin in combination with ascorbic acid regulates the severity of Staphylococcus aureus infection-induced septic arthritis in mice.

To study the effects of gentamicin in combination with ascorbic acid on septic arthritis, mice were infected with Staphylococcus aureus (S. aureus) and treated with gentamicin, which was given at 5 mg/kg after 24 h of infection, followed by ascorbic acid, given at 20 mg/kg body weight after 2 h of gentamicin treatment. Mice were sacrificed at 3, 9, 15 days post-infection (dpi). Combined treatment of infected mice with gentamicin and ascorbic acid eradicated the bacteria from the blood, spleen and synovial tissue and showed a significant gross reduction in arthritis, reduced serum levels of tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). S. aureus-infected mice have demonstrated the disturbed antioxidant status measured in terms of cellular antioxidants like reduced glutathione and antioxidant enzymes such as superoxide dismutase (SOD) and catalase. The same were ameliorated when the animals were co-treated with gentamicin along with ascorbic acid.

Intracellular survival of Staphylococcus aureus: correlating production of catalase and superoxide dismutase with levels of inflammatory cytokines.

BACKGROUND: Superoxide dismutase (SOD) and catalase are anti-oxidant enzymes potentially used by the bacteria to neutralize macrophage microbicidal molecules such as hydrogen peroxide (H2O2). OBJECTIVE: To investigate contribution of bacterial anti-oxidant enzymes in intracellular survival of Staphylococcus aureus (S. aureus) within macrophages. MATERIALS: Murine peritoneal macrophages and S. aureus (CMC-524, ICH-629 and ICH-757). TREATMENT: 10(6) colony forming units (CFU) of the 90 minutes (min) intracellularly viable S. aureus were administered (i.v.) per mouse through 0.1 ml saline. METHODS: Anti-oxidant enzyme assay, phagocytic activity, H2O2 release, Zymography for catalase, serum tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) level were estimated. One-way Model I ANOVA and one tail Student's t-test were performed. RESULTS: Survival of S. aureus was least after 90 min of reincubation within macrophages. Maximum amount of bacterial anti-oxidant enzymes were released after 90 min of re-incubation. H2O2 released after 90 min of re-incubation with S. aureus was maximum. Higher activity of catalase and SOD by S. aureus occurred in response to the gradual production of H2O2. Serum IL-6 and TNF-alpha was also elevated 1h post infection. CONCLUSIONS: Bacterial catalase and SOD combat reactive oxygen species enabling S. aureus to persist within macrophages, inducing local inflammation, causing greater induction of serum TNF-alpha and IL-6.

Synergism in immunotoxicological effects due to repeated combined administration of arsenic and lead in mice.

Arsenic and lead are considered potent human hazards because of their neoplastic outcomes; increasing epidemiologic evidence indicates a link between heavy metal exposure and health risk. Since health risks of singly administered metals are well-established, in the present study we determined whether simultaneous repeated multimetal (arsenic + lead) exposure influences the development of immunotoxicity in mice exposed (in vivo) to lead acetate (10 mg/kg b.w.) and sodium arsenite (0.5 mg/kg b.w.) simultaneously. We report that in vivo multimetal exposure alters cell morphology, inhibits cell adhesion, nitric oxide release, intracellular killing ability, chemotactic migration, myeloperoxidase release, bacterial clearance from blood and spleen and increases DNA fragmentation. On measuring bacterial density in blood and spleen after 0, 24, 48 and 72 h post infection (with Staphylococcus aureus MC524) in control and multimetal treated groups, bacterial load showed delayed clearance from blood and spleen in the multimetal exposed group. We also found that in vivo exposure to the multimetal caused a decrease in cell adhesion, indicated by a fall in absorbance at 570 nm with respect to control. Exposure to multimetal led to morphological changes in macrophages, since more deformed cells were obtained in repeated combined exposure to arsenic and lead compared to control. Nitric oxide, which has a potent microbicidal activity in macrophages, was found to be released in fewer amounts in the multimetal exposed group from that of control group. It was observed that the viability of bacteria gradually decreased in control macrophage with time, whereas, in macrophages of multimetal exposed mice, the viability of S. aureus gradually increased. Chemotactic migration of splenic macrophages significantly decreased in the multimetal exposed group from that of control. Lysosomal enzyme release from splenic macrophages decreased upon simultaneous exposure to arsenic and lead, as is evident from the decrease in myeloperoxidase release in multimetal group from that in control. That the structural integrity of splenic macrophages is decreased in the multimetal exposed group is also evident from the enhanced percentage of DNA fragmentation after multimetal exposure, suggesting apoptotic death of splenic macrophage. Intracellular viable bacteria in the splenic macrophage from multimetal exposed group was 89.16 +/- 3.54% while that from control group was 49.19 +/- 1.16%, whereas single metal exposed groups showed a bacterial viability of 69.6 +/- 2.45% and 71.71 +/- 1.89% in arsenic and lead treated groups respectively. What is essentially noteworthy from the observed results is that lead and arsenic causes a greater immunotoxic effect when administered together as multimetal than when singly administered. Simultaneous exposure to lead and arsenic appears to be additive as is further established from the isobologram constructed by plotting the concentration of arsenic against the concentration of lead at which effect (in this case myeloperoxidase release) remained constant, a convex line showing synergism was demonstrated. The present study reports a definite synergistic trend of immunotoxicity during simultaneous exposure to arsenic and lead, that is, a multimetal challenge, as compared to the effects of independent exposure to them.

Lead induced modulation of splenic macrophage responses on humoral and cell mediated immunity.

The heavy metal lead is an environmental toxic material that can induce pathophysiological changes in many organ systems. Previous studies have shown the effects of lead exposure on immune cells in different experimental animals, however, the mechanism of their influence on the immune system is unclear. We reported that in vivo lead exposure inhibits phagocytosis, nitric oxide release, induces DNA fragmentation suggesting the apoptotic death of the target cell. We have also presented evidence that inhibition of macrophage functional responses implicated alteration of humoral and cell mediated immunity. In vivo exposure to lead acetate alters the phagocytic capacity of splenic macrophages as evident from the reduction of phagocytic index of control from 19,792+/-1385.69 to 8893+/-893 in the treated group. The amount of nitric oxide released by the control cell 2.25+/-0.125 microM is also reduced to 1.9375+/-0.0625 microM upon in vivo lead treatment. Functional integrity of the target cell is also decreased after lead exposure as obtained from the percentage of DNA fragmentation. Control group shows 33.29+/-0.11% of fragmented DNA, which is enhanced to 42.43+/-0.725% following the lead treatment. A greater percentage of DNA fragmentation upon lead treatment probably indicating that the heavy metal induces apoptosis. The humoral immune response is also altered after lead exposure as indicated by the decrease of the antibody titre in control group from 1:2048 to 1:128 in the treated group. From the DTH reaction, it was observed that the mean diameter of swollen foot pad of control mice is 0.329+/-0.15 cm and that of lead treated mice is 0.274+/-0.056 cm. It can, therefore, be suggested that lead inhibits normal functional activities of splenic leukocytes, particularly phagocytosis and also affects the functional integrity of cells by inducing DNA fragmentation. The study may demonstrate the usefulness of investigation of humoral immune system and leukocyte functions as sensitive parameters in detecting the effects of lead toxicity.

Metabolic and immunological responses associated with in vivo glucocorticoid depletion by adrenalectomy in mature Swiss albino rats.

The study is undertaken to determine the effect of adrenal corticosteroid depletion after adrenalectomy on carbohydrate, protein and fat metabolism as well as maturation and functional efficacy of the immunocompetent cells. Beside biochemical and hematological parameters, whether in vivo glucocorticoid depletion has any modulatory effects on splenic macrophage responses to bacterial challenge with regards to intracellular killing, nitric oxide release and cellular integrity, were determined. Major findings of our study indicate that blood glucose, urea and total inorganic phosphate levels showed a time dependent increase in adrenalectomized rats compared to control. Total glycogen content in liver was decreased gradually due to adrenal corticosteroid insufficiency. Hematological parameters like hemoglobin concentration, hematocrit value, total leukocyte count and differential count were also found to increase in the adrenalectomized group with respect to intact group. From the functional study of immunocompetent cells, intracellular killing capacity of splenic macrophages recovered from control and adrenalectomized rats after 10 and 20 days of adrenalectomy showed no significant alteration; however, the function of splenic macrophages recovered from rats after 30 days of adrenalectomy showed altered response. Nitric oxide released from splenic macrophages of adrenalectomized rats was less than that of control animal even after stimulation with lipopolysaccharide. DNA fragmentation assay showed a lesser degree of fragmentation of splenic macrophages obtained from adrenalectomized rats indicating, apoptotic death of cells in this group decreases. Adrenal corticosteroid insufficiency due to adrenalectomy interferes with metabolic and hematopoietic functions and modulates the development and maintenance of normal immunitary status, which in turn influences the inflammatory response.

Effect of adrenalectomy on rat peritoneal macrophage response.

Glucocorticoid hormones are important for vital functions and act to modulate inflammatory and immune responses. In contrast to other hormonal systems no endogenous mediators have been identified that can directly counter-regulate their potent anti-inflammatory and immunosuppressive properties. Glucocorticoids are known to interfere with the ability of the macrophage not only to induce and amplify an immune response but also to inhibit macrophage inflammatory effector functions. Although the actual immunocompetence of animals undergoing endocrine gland ectomy has never been directly studied, there is no doubt that adrenal hormones are deeply involved in the development and maintenance of the immunitory functions and this may in turn influence the inflammatory reaction. To study the effect of endogenous glucocorticoids on the functions of rat peritoneal macrophages and induction of humoral immune response we observed some of the rat peritoneal macrophage effector functions, provided that endogenous glucocorticoids are depicted by adrenalectomy. The mean phagocytic index (PI) of control macrophage (Mphi) is increased from 23,825 +/- 427 to 31,895 +/- 83 after adrenalectomy (P < or = 0.001). Intracellular killing capacity in control cell is 82% which is found to be 73% in case of adrenalectomised cell (p < 0.05). The amount of nitric oxide released from control Mphi 20.25 +/- 1 microM following adrenalectomy shows the amount of nitric oxide release was 18.25 microM (p < or = 0.01 ). The percentage of DNA fragmentation in control Mphi was 68.82 +/- 4 which was reduced to 56.76 +/- 1 after adrenalectomy (p < or = 0.01). In sheep red blood cell (SRBC) immunised and adrenalectomised animal, agglutination titre was obtained at lowest antibody concentration (1 : 128) whereas serum from SRBC immunised normal rats showed early agglutination (1: 32). Endogenous glucocorticoid depleted rats show enhanced phagocytic capacity, antibody raising capacity as well as on the other hand adrenal hormone insufficiency reduces the intracellular killing capacity, nitric oxide (NO) release, improper cell maturation and heightens the probability of infection. These observations demonstrate a counter-regulatory system via glucocorticoid that functions to control inflammatory and immune responses.

Identification and characterization of specific receptor for interleukin-8 from the surface of human monocytes.

A specific receptor for interleukin-8 has been identified on the surface of human monocytes using 125I IL-8 as a probe. A binding kinetic pattern shows that saturation was attained after 90 min and that the receptor was distinct from the receptors of other cytokines (IL-L alpha, IL-2, TNF alpha, GMCSF) and FMLP. Scatchard analysis of the binding data shows that 7000-10,000 receptors /monocyte are present with an equilibrium Kd 7 x 10(-9) M. By immunoblot, the receptor for IL-8 showed a sharp band with approximate M.W. 59 kD, consistent with the M.W. of IL-8 receptor of neutrophils. In Boyden Chamber, monocytes migrated towards IL-8 and the cytokine was observed to induce transient rise of intracellular Ca+2 in the cells. Thus, identification of functionally active IL-8 receptor in monocyte may be helpful for understanding its possible role during inflammation.