Recombinant Murine Interferon-gamma Research Articles

Abstract 2994: Hypoxia blocks the presentation of tumor antigens to CD8+ T cells

Abstract Tumor hypoxia is known to enhance tumor growth and immune evasion by activating angiogenic pathways supporting tumor progression and fostering suppressive phenotypes of immune cells within the tumor-microenvironment. However, the consequence of tumor hypoxia on antigen processing and presentation to surveilling CD8+ T cells is a notable knowledge gap with direct relevance towards adaptive immune responses against cancer. Here, we present evidence using the well-established OT-I transgenic model that hypoxia decreases antigen specific T cell effector responses, and importantly, that these decreases in effector responses are not attributable to any hypoxic influence on T cell function. Murine B16 melanoma cells expressing ovalbumin (OVA; MO4) or parental B16 were grown under 20% or 2% oxygen for 48 hours. These cells were then treated with 0 or 10 ng/mL recombinant murine interferon gamma (IFN-g) for 24 hours at their respective oxygen conditions. For analysis of cytotoxicity and effector responses, these cells were labeled with a fluorescence enhancing ligand (BATDA) and then incubated with OT-I T cells recognizing OVA-peptide (SIINFEKL) presented by C57BL/6 mouse MHC I (H2kb) for 4 hours at 20% oxygen. Supernatants were then tested for BATDA release using a europium solution and subsequent fluorescent signal acquisition. OT-I T cells displayed significantly more specific lysis of target MO4 cells cultured under 20% oxygen compared to those cultured under 2% oxygen (p value < 0.0001; 2-way ANOVA). Control experiments using parental B16 target cells displayed little to no specific lysis. Additionally, OT-I T cells were incubated with B16 or MO4 cells cultured as described above for 18 hours under 20% oxygen in the presence of protein transport inhibitors and analyzed for effector cytokine production. Tumor necrosis factor alpha (TNF-a) production was significantly increased in OT-I cells co-cultured with IFN-g treated MO4 target cells preconditioned in both 20% and 2% oxygen (p value < 0.05; unpaired T test), however the fold change in TNF-a production for OT-I cells incubated with 20% oxygen cultured MO4 cells was approximately twice the increase observed in OT-I cells incubated with 2% oxygen cultured MO4 cells. These differences are not attributable to altered OVA expression under hypoxia. Furthermore, IFN-g dependent up-regulation of MHC-I and immunoproteasome catalytic subunits was found to be blocked by hypoxia, suggesting that hypoxic attenuation of the MHC I pathway is responsible for observed decreases in cytotoxic and effector responses in OT-I co-culture assays. Overall, these studies suggest that tumor hypoxia blunts anti-tumor immune responses irrespective of the oxygenation status of surveilling CD8+ T cells. Citation Format: Matthew G. Smith, Heena Panchal, Adam Mailloux. Hypoxia blocks the presentation of tumor antigens to CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2994.

IL-10 down-regulates the expression of survival associated gene hspX of Mycobacterium tuberculosis in murine macrophage

Mycobacterium tuberculosis (MTB) adopts a special survival strategy to overcome the killing mechanism(s) of host immune system. Amongst the many known factors, small heat shock protein 16.3 (sHSP16.3) of MTB encoded by gene hspX has been reported to be critical for the survival of MTB. In the present study, the effect of recombinant murine interferon-gamma (rmIFN-γ) and recombinant murine interleukin-10 (rmIL-10) on the expression of gene hspX of MTB in murine macrophage RAW264.7 has been investigated. By real-time RT-PCR, it was observed that three increasing concentrations (5, 25 and 50ng/ml) of rmIFN-γ significantly up-regulated the expression of hspX whereas similar concentrations of rmIL-10 (5, 25 and 50ng/ml) significantly down-regulated the hspX expression. This effect was not only dependent on the concentration of the stimulus but this was time-dependent as well. A contrasting pattern of hspX expression was observed against combinations of two different concentrations of rmIFN-γ and rmIL-10. The study results suggest that rIL-10 mediated down-regulation of hspX expression, in the presence of low concentration of rIFN-γ, could be used as an important strategy to decrease the dormancy of MTB in its host and thus making MTB susceptible to the standard anti-mycobacterial therapy used for treating tuberculosis. However, as these are only preliminary results in the murine cell line model, this hypothesis needs to be first validated in human cell lines and subsequently in animal models mimicking the latent infection using clinical isolates of MTB before considering the development of modified regimens for humans.

Sensitivity of insulin production from encapsulated islets to endotoxin-stimulated macrophage inflammatory mediators

Chronic inflammation may compromise function of implanted encapsulated islets. Increased purity of alginate used for encapsulation prolongs encapsulated graft function, correlating with decreased presence of impurities like bacterial endotoxin. Limits for endotoxin contamination in biomaterials based on indirect inhibition of function of embedded cells have yet to be established. In a coculture system with RAW 264.7 monocyte/macrophage cells in the presence of 50 ng/mL murine recombinant gamma-interferon (mrIFN-gamma), the insulin response to glucose challenge of both rat and pig unencapsulated islets was prevented by endotoxin (LPS) in the medium down to 0.3 EU/mL (LOEL), but not 0.06 EU/mL (NOEL). Evaluation of nitrite concentrations in supernatants revealed that pig islets were more resistant to LPS-stimulated macrophage mediators than rat islets. Encapsulation in highly purified alginate produced little change in observed inhibitory effects of macrophage-generated nitric oxide (NO) toward islet function. Chemically released NO was much less effective in inhibiting insulin responsiveness to glucose challenge than was coculture of islets with LPS and mrIFN-gamma-stimulated RAW 264.7. These results taken together with other data suggest that an upper limit of 0.3 EU/mL LPS within the encapsulating alginate will not impair the function of implanted encapsulated islets by toxic concentrations of macrophage-mediated inflammatory agents.

Upregulation of indoleamine 2,3‐dioxygenase in hepatocyte during acute hepatitis caused by hepatitis B virus‐specific cytotoxic T lymphocytes in vivo

Indoleamine-2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme inducing suppression of T-cell function and immune tolerance. In hepatitis B virus (HBV) transgenic (Tg) mice, the adoptive transfer of HBV-specific cytotoxic T lymphocytes (CTL) causes a necroinflammatory liver disease that is histologically similar to acute viral hepatitis in man. The present study aimed to determine IDO expression in the liver and hepatocytes during an acute hepatitis model. Serum l-kynurenine (l-Kyn) concentration in HBV Tg mice administered with HBV-specific CTL was measured over time, together with serum levels of alanine aminotransferase (ALT). Furthermore, we examined the expression of IDO in the total liver and isolated hepatocytes of HBV Tg mice after CTL injection using immunohistochemical analysis and reverse-transcription polymerase chain reaction (PCR). In HBV Tg mice, HBV-specific CTL induced, over the course of several days, a chronic increase in serum l-Kyn levels, which was associated with a sustained enhancement of liver IDO activity. In particular, IDO expression was enhanced in the liver parenchymal cells (hepatocytes) after HBV-specific CTL injection both in immunohistochemical analysis and in reverse-transcription PCR. Moreover, murine recombinant interferon-gamma (IFN-gamma) directly increased the IDO expression in primary hepatocytes in vitro. Cytotoxic T lymphocytes transduction results in the upregulation of IDO, which might downregulate T-cell responsiveness. Our findings provide evidence that hepatocyte itself expresses IDO and increases levels of l-Kyn in the blood in acute lethal hepatitis of mice. These data indicate that HBV infection facilitates the induction of IDO in response to proinflammatory cytokines, particularly IFN-gamma.

The effect of treatment with interferon-gamma on type II collagen-induced arthritis

We have investigated the effects of recombinant murine interferon-gamma (rIFN-gamma) on type II collagen-induced arthritis (CA) in DBA/l mice. Therapeutic as well as prophylactic treatment with subcutaneous rIFN-gamma, at 10(5) U/mouse six times a week, inhibited the development of CA without any obvious side effects. The accompanying suppression of anti-CII antibody responses may partly explain the inhibition of CA by rIFN-gamma. The possible role of the anti-inflammatory effect of systemic IFN-gamma in the inhibition of CA is discussed.

Screening biomaterials for stimulation of nitric oxide‐mediated inflammation

Inflammatory reactions to biomaterials may include macrophage-mediated generation of nitric oxide (NO), which may harm patient tissue or potentially interfere with proper function of an implanted device. RAW 264.7 cells were grown in culture and treated at various times with lipopolysaccharide (LPS, endotoxin), murine recombinant gamma-interferon (mrIFN-gamma), and different preparations of hyaluronic acid (HA). Increase in fluorescence of 2,3-diaminonaphthalene (DAN) allowed for detection of initial (24 h or less) NO inflammatory responses of RAW 264.7 to LPS from E. coli O26:B6. By looking at early time points, mrIFN-gamma augmentation of the LPS effect was observed, simulating a complex immune reaction. Activation through nuclear factor-kappaB (NF-kappaB), was confirmed in this system by parthenolide inhibition of LPS stimulation. Stimulation of RAW 264.7 by different HA preparations resulted in NO responses that correlated with the amount of LPS present. In the presence of mrIFN-gamma, a significant inflammatory reaction to HA was observed when the concentration of contaminating LPS was as low as 0.15 EU/mL. NO production in the presence of mrIFN-gamma by RAW 264.7 may serve as a convenient in vitro system to routinely screen biomaterials for potentially harmful macrophage-mediated inflammation whereby the safety of implanted medical devices might be compromised.

Failure to induce alopecia areata in C3H/HeJ mice with exogenous interferon gamma

Alopecia areata is a cell mediated autoimmune disease that targets actively growing, anagen stage hair follicles in several mammalian species. Upregulation of MHC I due to interferon gamma is considered to be one of the initiating steps. To test this hypothesis we used the spontaneous C3H/HeJ mouse model, induced anagen by wax stripping the skin, and injected recombinant murine interferon gamma. Alopecia areata is a complex polygenic trait with low penetrance in these mice. Injection of interferon gamma did not change the frequency or time of onset of alopecia in these mice suggesting this protein alone is not sufficient to initiate disease.

SHP-1 inhibits LPS-mediated TNF and iNOS production in murine macrophages

Several lines of evidence have suggested that protein tyrosine phosphatases, including CD45 and SHP-1, regulate macrophage activation. Macrophages from mice lacking SHP-1 (motheaten mice) are hyper-responsive to many stimuli, suggesting that SHP-1 may negatively regulate macrophage activation. Herein we report that the repressible/inducible over-expression of wild-type SHP-1 in a subclone of RAW 264.7 macrophages (RAW-TT10 cells) inhibited both TNF secretion and iNOS protein accumulation in response to stimulation with lipopolysaccharide (LPS) and recombinant murine interferon-gamma and led to diminished LPS-mediated tyrosine phosphorylation of vav1. In contrast, expression of a truncated SHP-1 construct previously shown to interfere with endogenous SHP-1 function modestly augmented LPS-mediated TNF and iNOS production and did not inhibit vav1 tyrosine phosphorylation. Taken together, these data provide the first direct evidence that SHP-1 inhibits macrophage activation by LPS and suggest that this effect may be mediated in part by dephosphorylation of vav1.

Effect of nitric oxide on the growth ofChlamydophila pneumoniae

Chlamydophila pneumoniae is an important human intracellular pathogen; however, the pathogenesis of C. pneumoniae infection is poorly understood and the immune control mechanism versus host cells is not completely known. The role of the nitric oxide (NO) synthase pathway in inhibiting the ability of C. pneumoniae to infect macrophage J774 cells and the ability of NO to damage isolated C. pneumoniae were investigated. Exposure of infected cultures to recombinant murine gamma interferon (MurIFN-gamma) resulted in increased production of NO and reduced viability. Addition of 2-(N,N-diethylamino)-diazenolase-2-oxide before infection of J774 cells or during chlamydial cultivation released NO, both resulting in a reduction in the viability of C. pneumoniae in a dose-dependent way. These results indicate that immune control of chlamydial growth in murine macrophage cells may trigger a mechanism that includes NO release with effects on the multiplication of the microorganism, thus suggesting that NO may play a role in preventing the systemic spread of Chlamydia.

Transfection of the genes for interleukin-12 into the K1735 melanoma and the EMT6 mammary sarcoma murine cell lines reveals distinct mechanisms of antitumor activity.

Interleukin 12 (IL-12) is a pleiotropic cytokine with multiple effects on the immune system. The antitumor effects of locally produced IL-12 were examined in 2 tumor model systems. IL-12 expressing EMT6 mammary sarcomas (EMT6/IL-12) grew temporarily and then regressed resulting in mice that were immune to a further challenge of EMT6 cells. Interestingly, the IL-12 expressing K1735 melanomas (K1735/IL-12) maintained a lag phase of nonmeasurable growth for several weeks, followed by tumor outgrowth that was associated with a loss of IL-12 production. Tumor-infiltrating lymphocytes (TILs) isolated from EMT6/IL-12 tumors effectively lysed EMT6 target cells, whereas K1735/IL-12 TILs lacked lytic activity. Both IL-12 expressing tumors, however, grew progressively in nude mice indicating an important role for T cells in each case. Recombinant murine interferon gamma (rmIFN-gamma) inhibited the growth of EMT6 cells, but not K1735 cells in vitro, and strongly induced the expression of the antiangiogenic chemokine interferon-inducible protein 10 (IP-10) by both cell lines. Of interest, only the EMT6 cell line was able to secrete the proangiogenic molecule, vascular endothelial growth factor (VEGF), in response to low oxygen conditions. Fluorescent staining of the vascular endothelium at the tumor injection site provided images depicting early stages of angiogenesis prior to K1735/IL-12 tumor outgrowth. These results indicate that locally produced IL-12 likely mediates the rejection of EMT6 tumors through tumor cell lysis by host immune cells, whereas its antiangiogenic potential may be counterbalanced by the strong induction of VEGF by hypoxic tumor cells. In contrast, IL-12 does not induce protective immunity to K1735 tumors. However, an antiangiogenic mechanism may be responsible for controlling tumor growth.

Type-1 transforming growth factor-beta differentially modulates tumoricidal activity of murine peritoneal macrophages against metastatic variants of the B16 murine melanoma.

Transforming growth factor-beta 1 (TGF-beta 1) renders mouse peritoneal macrophages tumoricidal against metastatic variants of the B16 mouse melanoma in vitro. Both direct cytotoxicity and indirect cytotoxicity were observed. A subthreshold concentration (10 U/ml) of recombinant murine interferon-gamma (rMuIFN-gamma) enhanced the direct tumoricidal activity of TGF-beta 1-activated macrophages from 29% to 88% but did not change their indirect tumoricidal profile. Data obtained from macrophages preincubated with either TGF-beta 1 or rMuIFN-gamma showed that TGF-b1 can initiate tumoricidal activity better than rMuIFN-gamma. These effects were plasma-membrane mediated because targeting macrophages with liposomal TGF-beta 1 was ineffective. The order of tumoricidal susceptibility of the B16 melanoma lines to activated macrophages was B16F1 > B16F10 > B16BL6, in inverse order of metastatic potential.

Effect of interleukin-18 on mouse core body temperature.

We have studied, using a telemetry system, the pyrogenic properties of recombinant murine interleukin-18 (rmIL-18) injected into the peritoneum of C57BL/6 mice. The effect of IL-18 was compared with the febrile response induced by human IL-1beta, lipopolysaccharide (LPS), and recombinant murine interferon-gamma (rmIFN-gamma). Both IL-1beta and LPS induced a febrile response within the first hour after the intraperitoneal injection, whereas rmIL-18 (10-200 microg/kg) and rmIFN-gamma (10-150 microg/kg) did not cause significant changes in the core body temperature of mice. Surprisingly, increasing doses of IL-18, injected intraperitoneally 30 min before IL-1beta, significantly reduced the IL-1beta-induced fever response. In contrast, the same pretreatment with IL-18 did not modify the febrile response induced by LPS. IFN-gamma does not seem to play a role in the IL-18-mediated attenuation of IL-1beta-induced fever. In fact, there was no elevation of IFN-gamma in the serum of mice treated with IL-18, and a pretreatment with IFN-gamma did not modify the fever response induced by IL-1beta. We conclude that IL-18 is not pyrogenic when injected intraperitoneally in C57BL/6 mice. Furthermore, a pretreatment with IL-18, 30 min before IL-1beta, attenuates the febrile response induced by IL-1beta.

Effect of administration of a recombinant adenovirus expressing the genes for IFN-gamma and interleukin-12 on acute murine toxoplasmosis.

The effect of recombinant murine interferon-gamma (rMuIFN-gamma) produced from an adenovirus construct on Toxoplasma gondii in tissue culture and on the outcome of a T. gondii infection in mice was determined. Supernatants from AdCMVMuIFN-gamma-infected mouse lung epithelial (MuLE) cells were evaluated for the ability to produce biologically active IFN-gamma by measuring the capacity of the supernatants to activate peritoneal macrophages for killing of T. gondii. The bioactivity of IFN-gamma in supernatants increased with increasing multiplicity of infection (moi). Replication was inhibited 43%, 67%, and 70% by supernatants from MuLE cells infected with AdCMVMuIFN-gamma moi 5, 10, and 50, respectively, (p < 0.01 compared with controls). Bioactivity of IFN-gamma also increased as the length of time after infection increased. T. gondii replication was inhibited 28% and 36%, respectively, by AdCMVMuIFN-gamma-infected MuLE cell supernatants recovered at 24 and 48 h (p < 0.01 compared with control). In vivo administration of AdCMVMuIFN-gamma exhibited 33% mortality by day 9 in mice acutely infected with T. gondii compared with 100% mortality in control mice (p = 0.045). Administration of AdCMVIL-12 reduced mortality to 40% compared with control mice. However, this reduction was not significant (p = 0.08). Overall survival was extended 2 days with AdCMVMuINF-gamma administration and 5 days with AdCMVIL-12. AdCMVMuIFN-gamma in vitro inhibits T. gondii, and in vivo AdCMVMuIFN-gamma and AdCMVIL-12 lead to increased survival in mice.

Arbortristosides Modulate Murine Peritoneal Macrophages For Phagocytosis And Intracellular Killing Of Candida Albicans

Studies indicate that arbortristoside C (50 µg/ml)-primed murine peritoneal macrophages (MuPMØs) provide high phagocytosis (85%) in a concentration-dependent manner at a target: effector ratio of 1:5, with an optimal Phagocytic Index (PI) of 8.2 at 6 hr, compared to arbortristoside A or control. Recombinant murine interferon gamma (rMuIFN-?)-primed MuPMØ, at 100 U, provided 98% phagocytosis with a PI 8.5 at 24 hr. The intracellular killing of C. albicans in arbortristoside C-primed MuPMØ was enhanced to 10% at 1 hr, and 25% at 3 hr, compared to control. The rMuIFN-?-primed MuPMØ, at 100 U, yielded 50% killing of blastospores at 3 hr, compared to 10 U of rMuIFN-? which was less detrimental (45%) in a concentration- and time-dependent manner. The intracellular residence of blastospores beyond 3 hr caused rupturing and killing of MuPMØs due to extensive germ tube formation. Transmission electron microscopic evidence further indicates arbortristoside C-primed MuPMØ activated lysosomal machinery causes fusion of phagolysosome with digested blastospores.

Cyclosporin A and tacrolimus (FK506) suppress expression of inducible nitric oxide synthase in vitro by different mechanisms.

1 The effects of the immunosuppressant drugs cyclosporin A and tacrolimus (FK506) on nitric oxide synthesis were examined in a murine macrophage cell line (J774) and rat vascular smooth muscle cells (VSMC) in culture for 24 and 48 h, respectively. 2 Cyclosporin A (0.01-10 microM) inhibited by up to 90% accumulation of nitrite induced by lipopolysaccharide (LPS) in both cell lines, but FK506 (0.01-10 microM) had a weaker effect on nitrite accumulation in these cells. Cyclosporin A and FK506 (at 1 microM) also significantly inhibited nitrite production induced by recombinant murine interferon-gamma (rIFNgamma) and recombinant murine interleukin-1beta (rIL-1beta) in J774 and VSMC, respectively. 3 In J774 cells, cyclosporin A (but not FK506) at 1 microM was inhibitory when co-incubated with the inducing agents but not when the cells were treated with the immunosuppressant before or after the inducer. In VSMC, nitrite production was inhibited by co-incubation of cyclosporin A or FK506 with the inducer, or when the immunosuppressants were pre-incubated with cells. In contrast, N-monomethyl L-arginine (NMMA) abolished nitrite production when incubated with either cell type during or after addition of inducing agent, but not if cells were preincubated with NMMA. 4 RNA extracted from treated J774 and VSMC was subjected to reverse transcription-polymerase chain reaction (RT-PCR). Cyclosporin A, but not FK506, suppressed expression of mRNA for NOS2 in a concentration-dependent manner when co-incubated with LPS. 5 The fact that the potency difference between cyclosporin A and FK506 for NO suppression is the opposite to that for inhibition of interleukin-2 generation suggests that the immunosuppressants act in J774 macrophages and VSMC through intracellular mechanisms that differ from those elucidated in T-cells. Cyclosporin A suppresses NOS2 gene transcription, but FK506 acts post-transcriptionally to suppress NO generation in VSMC. 6 Taken together the present data suggest that therapeutic concentrations of cyclosporin A, but not FK506, might well suppress NO production, but FK506 would not have this effect. Suppression of NO might contribute to the side effects of hypertension and nephrotoxicity associated with long-term use of cyclosporin A to prevent transplant rejection.

Pneumococci stimulate the production of the inducible nitric oxide synthase and nitric oxide by murine macrophages.

The role of nitric oxide (NO) in the pathophysiology of gram-positive sepsis is uncertain. In inflammatory conditions, high-output NO production is catalyzed by the enzyme inducible nitric oxide synthase (iNOS). The ability of 2 strains of pneumococci, pneumococcal cell wall preparations, and purified pneumococcal capsule (Pnu-Imune 23) to trigger the production of iNOS protein and NO in RAW 264.7 murine macrophages was tested. Live pneumococci, oxacillin-killed pneumococci, and pneumococcal cell wall preparations stimulated the production of iNOS and NO by RAW 264.7 cells in the presence, but not the absence, of low concentrations of recombinant murine interferon-gamma. In contrast, purified pneumococcal capsule induced little or no iNOS or NO production by these cells. Thus, pneumococci stimulate high-output NO production by murine macrophages. The potential role of NO in the pathogenesis of pneumococcal sepsis deserves further study.

The role of cytokines in MET-enkephalin-modulated nitric oxide release.

In the present study the in vitro and in vivo effect of Met-enkephalin (MENK) on nitric oxide (NO) release by mouse peritoneal macrophages was evaluated. While in vitro MENK was ineffective unless combined with suboptimal concentrations of recombinant murine interferon gamma, in vivo all the doses (2.5, 5 or 10 mg/kg bw) bimodaly modulated NO release. Only the stimulative (2.5 and 10 mg/kg bw) and not the suppressive (5 mg/kg bw) dose of MENK was opioid receptor-mediated as demonstrated by abolishing the effect by naloxone. The stimulative effect of the low (2.5 mg/kg bw) dose, that was observed only if MENK was injected p.m., was associated with the IL production and IFN gamma as demonstrated by abolishing the effect by specific antibodies. The data additionally support the idea that opioid-mediated responses might be to a large degree mediated by the release of cytokines.

STREPTOCOCCAL PYROGENIC EXOTOXINS A (SpeA) AND C (SpeC) STIMULATE THE PRODUCTION OF INDUCIBLE NITRIC OXIDE SYNTHASE (iNOS) PROTEIN IN RAW 264.7 MACROPHAGES

Streptococcal pyrogenic exotoxins A (SpeA) and C (SpeC) are members of a family of superantigens produced by group A streptococci that appear to play a key role in the pathogenesis of streptococcal toxic shock syndrome. Since it is known that nitric oxide (NO) and tumor necrosis factor (TNF) are largely responsible for the shock and multiple organ dysfunction of Gram-negative sepsis, we hypothesized that SpeA and/or SpeC could trigger the production of inducible nitric oxide synthase (iNOS) and/or TNF by murine macrophages. We exposed RAW 264.7 macrophages to increasing concentrations of SpeA or SpeC alone and in combination with recombinant murine interferon-gamma (rIFNγ) for 16-24 h. We found that both SpeA and SpeC triggered iNOS production in the presence of low concentrations of rIFNγ, while neither provoked iNOS accumulation in the absence of rIFNγ. Neither SpeA nor SpeC (with or without rIFNγ) reproducibly induced TNF production by these murine macrophages. These data indicate that two streptococcal exotoxins up-regulate iNOS production by murine macrophages and suggest that nitric oxide production may play an important role in the pathogenesis of streptococcal toxic shock syndrome.

Interleukin-12 and -10 and gamma interferon regulate polyclonal and ligand-specific expansion of murine B-1 cells

B-1 cells (CD5+ B220+) are a self-replenishing lineage of B cells which are autoreactive and capable of producing large amounts of interleukin-10 (IL-10). In mice experimentally infected with the human helminth parasite Schistosoma mansoni, expansion of B-1 cells is seen in the peritoneal cavity just prior to egg laying. In naive mice, B-1 cell expansion can be elicited by intraperitoneal injection of saline soluble egg antigens (SEA) or the polylactosamine sugar lacto-N-fucopentaose III (LNFPIII), which contains the Lewis-X trisaccharide. In this study, we demonstrate that LNFPIII is the major stimulus in SEA responsible for expansion of B-1 cells, since SEA-induced B-1 outgrowth is blocked by multiple injections of non-cross-linked free LNFPIII. IL-10 is an autocrine growth factor for B-1 cells, and we show that B-1 outgrowth after SEA and LNFPIII administration is inhibited by injection of anti-IL-10 antibodies. Furthermore, SEA- and LNFPIII-induced expansion of B-1 cells is inhibited by in vivo administration of recombinant murine IL-12 or recombinant gamma interferon. These data suggest that activation and expansion of IL-10-producing B-1 cells are governed via cross-regulatory cytokines.

ACQUIRED HYPORESPONSIVENESS TO BACTERIAL LIPOPOLYSACCHARIDE AND INTERFERON-γ IN RAW 264.7 MACROPHAGES

Bacterial lipopolysaccharide (LPS) can induce hyporesponsiveness to its own toxic effects, as reflected by the diminished production of tumor necrosis factor (TNF) and other inflammatory mediators by animals (and by purified macrophages) upon re-challenge with LPS. We have examined the regulation of TNF and inducible nitric oxide synthase (iNOS) production in the murine macrophage cell line RAW 264.7. These cells accumulated TNF mRNA and secreted TNF protein in a dose-dependent fashion after exposure to either LPS or recombinant murine interferon-gamma (riFN-gamma). Pre-exposure of RAW 264.7 cells to relatively high concentrations of LPS (> 10 ng/mL) resulted in diminished production of TNF in response to subsequent challenge with either LPS or riFN-gamma. In contrast, production of iNOS mRNA was either unaffected or augmented by pre-exposure of these cells to LPS. Our findings indicate that the hyporesponsive state of RAW 264.7 macrophages preincubated with LPS is not specific for LPS and that the production of TNF and nitric oxide (NO) by macrophages are differentially regulated.