Local inflammation,lethality and cytokine release in mice injected with Bothrops atrox venom.

We have provided evidence that: (a) lethality of mice to crude Bothrops venom varies according the isogenic strain (A/J > C57Bl/6 > A/Sn > BALB/c > C3H/HePas > DBA/2 > C3H/He); (b)BALB/c mice (LD50=100.0 microg) were injected i.p. with 50 microg of venom produced IL-6, IL-10, INF-gamma, TNF-alpha and NO in the serum. In vitro the cells from the mice injected and challenged with the venom only released IL-10 while peritoneal macrophages released IL-10, INF-gamma and less amounts of IL-6; (c) establishment of local inflammation and necrosis induced by the venom, coincides with the peaks of TNF-alpha, IFN-gamma and NO and the damage was neutralized when the venom was incubated with a monoclonal antibody against a 60 kDa haemorrhagic factor. These results suggest that susceptibility to Bothrops atrox venom is genetically dependent but MHC independent; that IL-6, IL-10, TNF-alpha, IFN-gamma and NO can be involved in the mediation of tissue damage; and that the major venom component inducers of the lesions are haemorrhagins.     

Secretion of TNF-alpha, IL-8 and nitric oxide by macrophages activated with Agaricus blazei Murill fractions in vitro

Water extracts of the mycelial culture and fruiting bodies of Agaricus blazei Murill were fractionated by ethanol precipitation using various ethanol concentrations. Original water extracts from mycelia (Fraction A-0) and fruiting bodies (Fraction B-0) induced TNF-alpha secretion by macrophages derived from rat bone marrow. Fractions B-4 and B-5 obtained from ethanol precipitation of fruiting bodies using 44% and 50% ethanol, respectively, and Fraction B-6 obtained from the supernatant at 50% ethanol markedly induced TNF-alpha secretion. Similar effects were observed in IL-8 secretion by macrophages. Regarding nitric oxide (NO), Fraction B-5 induced a significant increase in NO secretion and Fractions B-4 and B-6 induced slightly NO secretion. Northern blot analysis showed that the increases in cytokine- and NO secretion were due to an increase in cytokine mRNAs or NO synthase mRNA. Therefore, it is concluded that Agaricus blazei Murill components which activate macrophages result in the induction of cytokine- and NO secretion in vitro.     

Cytokine and nitric oxide release by J774A.1 macrophages is not regulated by estradiol.

Estradiol is able to regulate the release of inflammatory mediators by macrophages; however, the presence, extent, and direction of this modulation varies with species, tissue of origin, and cell culture conditions. This study examines the effects of 17-beta-estradiol (E2) on the release of inflammatory mediators by the J774A.1 mouse macrophage cell line. For experiments, cells were plated in phenol red-free DMEM containing 5% charcoal-dextran stripped calf serum. Western analysis showed that J774A.1 cells contain the estrogen receptor alpha (ER alpha) protein. We found that physiological and pharmacological levels of E2 (10(-12) M-10(-6) M) have no effect on the release of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), or monocyte chemoattractant protein-1 (MCP-1). This suggests that J774A.1 cells grown under these culture conditions would be useful for the investigation of non-estrogen-dependent mechanisms by which certain endocrine disruptors may affect their targets in macrophages.     

Immunomodulatory effects of the Tityus serrulatus venom on murine macrophage functions in vitro

Tityus serrulatus scorpion venom (TSV) consists of a very complex mixture of molecules and demonstrates significant immunomodulatory activities capable of stimulating immune functions in vivo. The purpose of this study was to compare the crude TSV with fractionated toxins extracted from this venom in order to determine which toxin(s) presented immunomodulatory effects on peritoneal macrophages. TSV was fractionated using gel filtration chromatography resulting in 5 heterogeneous fractions. The effects of these different fractions were analysed in vitro using detection by means of cytokines, oxygen intermediate metabolites (H2O2), and nitric oxide (NO) in supernatants of peritoneal macrophages. Several functional bioassays were employed: tumor necrosis factor (TNF) activity was assayed by measuring its cytotoxic activity in L929 cells, and other cytokines were assayed by enzyme-linked immunosorbent assay, whereas NO levels were detected by Griess colorimetric reactions in culture supernatant of macrophages exposed to different fractions. In vitro studies revealed that all fractions studied here presented an increment in H2O2, NO , and cytokines levels. The more pronounced increments were observed in macrophage cultures exposed to fraction FII which demonstrated that (a) the highest levels of IL-1alpha, IL-beta, and TNF were observed after 12 hours and that (b) the maximum levels of IFN-gamma and NO were observed after 72 hours. Taken together, these data indicate that fractions have a differential immunomodulating effect on macrophage secretion, and that FII is a potent activator of TNF production of macrophages.     

Modulation of macrophage phenotype by soluble product(s) released from neutrophils

The regulation of macrophage phenotype by neutrophils was studied in the s.c. polyvinyl alcohol sponge wound model in mice made neutropenic by anti-Gr-1 Ab, as well as in cell culture. Wounds in neutropenic mice contained 100-fold fewer neutrophils than those in nonneutropenic controls 1 day after sponge implantation. Wound fluids from neutropenic mice contained 68% more TNF-alpha, 168% more IL-6, and 61% less TGF-beta1 than those from controls. Wound fluid IL-10 was not different between the two groups, and IL-4 was not detected. Intracellular TNF-alpha staining was greater in cells isolated from neutropenic wounds than in those from control wounds. The hypothesis that wound neutrophil products modulate macrophage phenotype was tested in Transwell cocultures of LPS-stimulated J774A.1 macrophages and day 1 wound cells (84% neutrophils/15% macrophages). Overnight cocultures accumulated 60% less TNF-alpha and IL-6 than cultures of J774A.1 alone. The suppression of cytokine release was mediated by a soluble factor(s), because culture supernatants from wound cells inhibited TNF-alpha and IL-6 release from LPS-stimulated J774A.1 cells. Culture supernatants from purified wound neutrophils equally suppressed TNF-alpha release from LPS-stimulated J774A.1 cells. Wound cell supernatants also suppressed TNF-alpha and superoxide release from murine peritoneal macrophages. The TNF-alpha inhibitory factor has a molecular mass <3000 Da and is neither PGE2 nor adenosine. The present findings confirm a role for neutrophils in the regulation of innate immune responses through modulation of macrophage phenotype.     

Outer membrane protein A (OmpA) binds to and activates human macrophages

Outer membrane protein (Omp)A is highly represented and conserved in the Enterobacteriaceae family. Using a recombinant OmpA from Klebsiella pneumoniae (P40), we have analyzed the interaction between OmpA and macrophages. We report that Alexa488-labeled P40 binds (at 4 degrees C) to murine and human macrophages in a dose-dependent manner and is rapidly internalized (at 37 degrees C). No binding or internalization of the Alexa488-labeled glycophorin A control protein is observed under the same conditions. Furthermore, P40 up-regulates the production of IL-1beta, IL-8, IL-10, IL-12, and TNF-alpha by human macrophages and of NO by the RAW 264.7 murine macrophage cell line. P40 also synergizes with IFN-gamma and suboptimal concentrations of LPS to up-regulate the production of these mediators. In conclusion, P40 binds to and activates macrophages. These data suggest that recognition of OmpA by macrophages may be an initiating event in the antibacterial host response.     

Macrolide antibiotics protect against immune complex-induced lung injury in rats: role of nitric oxide from alveolar macrophages.

Macrolide antibiotics have unique immunomodulatory actions apart from antimicrobial properties. We studied the effects of macrolides on IgG immune complex (IgG-ICx)-induced lung injury in rats in vivo and in vitro. Intrapulmonary deposition of IgG-ICx produced a time-dependent increase in the concentration of NO in exhaled air. There were corresponding increases in the number of neutrophils accumulated into alveolar spaces, and lung wet-to-dry weight ratio. All of these changes were inhibited by pretreatment with erythromycin or josamycin, but not by amoxicillin or cephaclor. Incubation of cultured pulmonary alveolar macrophages caused up-regulation of NO production and expression of inducible NO synthase mRNA, an effect that was dose dependently inhibited by erythromycin, roxithromycin, or josamycin. The macrolides also reduced IgG-ICx-induced release of IL-1beta and TNF-alpha, but did not alter the release of NO induced by exogenously added IL-1beta and TNF-alpha. These results suggest that macrolide antibiotics specifically inhibit immune complex-induced lung injury presumably by inhibiting cytokine release and the resultant down-regulation of inducible NO synthase gene expression and NO production by rat pulmonary alveolar macrophages.     

Quantitative aspects of lipopolysaccharide and cytokine requirements to generate nitric oxide in macrophages from LPS-hyporesponsive (<Emphasis Type="Italic">Lps</Emphasis><Superscript>d</Superscript>) C3H/HeJ mice

Due to a gene defect (Lps(d)), C3H/HeJ mice are known to be hyporesponsive to the immunobiological potential of lipopolysaccharide (LPS). We studied dose requirements for LPS, IFN-gamma, and cytokines TNF-alpha and IL-10 to produce nitric oxide (NO) in peritoneal macrophages (Mphi) from these animals. In contrast to the Lps(n) C3H/HeN mice, high concentrations of LPS (up to 5 microg/mL) or IFN-gamma (up to 5 ng/mL) by themselves were unable to activate NO production in C3H/HeJ Mphi. The failure to produce NO could not be overcome by addition of L-arginine or tetrahydropterin. The high-output NO biosynthesis was dose-dependently stimulated by combined administration of varying concentrations of IFN-gamma (50-5000 pg/mL) and LPS (approximately 1 ng/mL) or to a lesser extent by IFN-gamma plus TNF-alpha or TNF-alpha/IL-10. Formation of NO in C3H/HeJ MCO triggered by high concentration of LPS (approximately 1 microg/mL) given together with IFN-gamma (0.2-5 ng/mL) reached the values typical for Lps(n) C3H/HeN mice. While Mphi from C3H/HeN mice secreted TNF-alpha, IL-10, and IL-10 upon contact with a low dose of LPS (1 ng/mL), C3H/HeJ Mphi required high concentration of LPS (5 microg/mL) to enhance the secretion of the cytokines. Yet, this dose remained ineffective to stimulate IFN-gamma in Mphi from C3H/HeJ mice. It can be presumed that one of the important factors influencing their deficient ability to form NO is a failure of Mphi to produce IFN-gamma upon LPS contact.     

IFN-gamma-independent autocrine cytokine regulatory mechanism in reprogramming of macrophage responses to bacterial lipopolysaccharide.

Macrophages are now well recognized to have a critical role in both innate and acquired immunity. The sentinel macrophage function is highly regulated and serves to allow for intrinsic plasticity of the innate immune responses to potential environmental signals. However, the mechanisms underlying the dynamic properties of the cellular arm of innate immunity are poorly understood. Therefore, we have conducted a series of in vitro studies to evaluate the contribution of immunoregulatory cytokines, such as IFN-gamma, IL-10, and IL-12, in modulation of macrophage responses. We found that macrophages from IFN-gamma knockout (IFN-gamma(-/-)) mice exhibit only marginal LPS-induced TNF-alpha, IL-12, and NO responses, all of which can be fully restored in the presence of rIFN-gamma. Pretreatment with substimulatory LPS concentrations led to reprogramming of IFN-gamma(-/-) macrophage responses in a dose-dependent manner that manifested by an increased TNF-alpha and IL-12, but not NO, production upon the subsequent LPS challenge. These reprogramming effects were substantially attenuated and profoundly enhanced in macrophages from IL-12(-/-) and IL-10(-/-) mice, respectively, as compared with those modulated in macrophages from the congenic wild-type mice. LPS-dependent reprogramming was also fully reproduced in macrophages isolated from SCID mice after immunodepletion of NK cells. Our data strongly imply that cytokine (TNF-alpha and IL-12), but not NO, responses in macrophages may, at least in part, be governed by an autocrine IFN-gamma-independent regulatory mechanism reciprocally controlled by IL-10 and IL-12. This mechanism may serve as an alternative/coherent pathway to the canonical IFN-gamma-dependent induction of antimicrobial and tumoricidal activity in macrophages.     

Nitric oxide synthesis and TNF-alpha secretion in RAW 264.7 macrophages: mode of action of a fermented papaya preparation

Macrophage inducible nitric oxide synthase is able to generate massive amounts of nitric oxide (NO) which contributes to the host immune defense against viruses and bacteria. Monocyte-macrophages stimulated with the bacterial wall component lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-gamma) express the inducible form of nitric oxide synthase (iNOS). Furthermore, tumor necrosis factor-alpha (TNF-alpha) is one of the central regulatory cytokines in macrophage antimicrobial activity and synergizes with IFN-gamma in the induction of NO synthesis. Because of its pivotal role in both antimicrobial and tumoricidal activities of macrophages, a significant effort has focused on developing therapeutic agents that regulate NO production. In the present study fermented papaya preparation (FPP) is shown to exert both immunomodulatory and antioxidant activity in the macrophage cell line RAW 264.7. Interestingly, a low and a high molecular weight fraction (LMF and HMF, respectively) of FPP exhibited different activity patterns. FPP fractions alone did not affect NO production. However in the presence of IFN-gamma, both LMF and HMF significantly increased iNOS activity and nitrite as well as nitrate accumulation. NO radical formation measured in real-time by electron paramagnetic resonance spectroscopy was higher in the presence of LMF and IFN-gamma. On the contrary, iNOS mRNA levels were enhanced further with HMF than with LMF. Moreover, LMF displayed a stronger superoxide anion scavenging activity than HMF. In the presence of IFN-gamma, both FPP fractions stimulated TNF-alpha secretion. However in non-stimulated macrophages, TNF-alpha secretion was enhanced by HMF only. Since water-soluble FPP fractions contained no lipid A, present data indicate that FPP is a macrophage activator which augments nitric oxide synthesis and TNF-alpha secretion independently of lipopolysaccharides.     

Effect of storage xyloglucans on peritoneal macrophages

Xyloglucans from seeds of Copaifera langsdorffii (XGC), Hymenaea courbaril (XGJ) and Mucuna sloanei (XGM) were obtained from milled and defatted cotyledons by aqueous extraction at 25 degrees C. The resulting fractions contained Glc, Xyl and Gal in molar ratios of 2.5: 1.5: 1.0 (XGC), 3.8: 2.6: 1.0 (XGJ) and 2.5: 1.6: 1.0 (XGM). HPSEC-MALLS/RI analysis showed that each polysaccharide fraction was homogeneous; M(w) values were 1.6 x 10(5), 2.0 x 10(5) and 1.5 x 10(5)g/mol, respectively. The effect of the xyloglucans on the production of O(2)*(-) and NO* and on the recruitment of macrophages to the mouse peritoneum was evaluated. All polysaccharides promoted an increase in the number of peritoneal macrophages in a dose-dependent manner. The largest increase, of 576% in comparison to the control group, was elicited by XGJ at 200 mg/kg. The effect of XGC, XGJ and XGM on O(2)*(-) production, in the presence or absence of phorbol 12-myristate 13-acetate (PMA), was not statistically significant. For NO(.) production, the lowest concentration of XGC (10 microg/ml) gave rise to an increase of 262% when compared to the control group; the effect was dose-dependent, reaching 307% at 50 microg/ml. On the other hand, XGJ at a concentration of 50 microg/ml enhanced NO* production by 92%. XGM did not affect NO* production significantly. The results indicate that xyloglucans from C. langsdorffii, H. courbaril and M. sloanei have immunomodulatory activity.     

Interleukin-10 (IL-10) inhibits the induction of nitric oxide synthase by interferon-gamma in murine macrophages.

A murine macrophage cell line, J774, expresses high levels of the enzyme nitric oxide synthase (NOS) and produces large amounts of nitric oxide (NO) when activated with recombinant interferon (IFN)-gamma and a low concentration of LPS (10 ng/ml). Both the expression of NOS and the production of NO were inhibited by recombinant IL-10 in a dose-dependent manner. The inhibition was effective only when the cells were pretreated with IL-10; addition of IL-10 at the same time or after IFN-gamma activation was without effect. These results demonstrate that IL-10, a product of Th2 (helper T lymphocyte 2) cells, can antagonise the function of IFN-gamma, a product of Th1 cells, by modulating the mechanism of synthesis of nitric oxide in the macrophages.