<Emphasis Type="Italic">In Vitro</Emphasis>, Melatonin Treatment Decreases Nitric Oxide Levels in Murine Splenocytes Cultured with the Venezuelan Equine Encephalomyelitis Virus

The purpose of this work was to determine the effect of melatonin on the nitric oxide levels in murine splenocytes cultured with the Venezuelan equine encephalomyelitis virus. After incubation, nitric oxide levels were measured by the diazotization assay. Those cultures with the Venezuelan equine encephalomyelitis virus increased nitric oxide levels. Splenocytes infected and treated with 100 and 150 μg/ml of melatonin, decreased significantly the nitric oxide levels when compared to infected and non-treated splenocytes. These findings show that splenocytes infected with the Venezuelan equine encephalomyelitis virus generate important amounts of nitric oxide and suggest that melatonin protects the mice infected with the Venezuelan equine encephalomyelitis virus by a mechanism involving the decreasing of nitric oxide concentrations in tissue.     

T cell substance P receptor governs antigen-elicited IFN-gamma production

Substance P (SP) enhances antigen-dependent T cell IFN-gamma production. It was determined if a T cell neurokinin-1 receptor (NK-1R) was critical for IFN-gamma regulation. T cells from schistosome-infected mice were mixed with splenocytes from uninfected NK-1R knockout (KO) animals. Thus only the schistosome egg antigen-specific T cells expressed NK-1R. The cells were cultured 18 h with or without SP. SP enhanced antigen-induced IFN-gamma production fourfold without affecting IL-4 or IL-5 secretion. NK-1R inhibitor blocked this stimulation. Neither purified T cells nor naive KO splenocytes cultured alone responded to antigen. To further define the importance of T cell NK-1R, we developed a T cell-selective NK-1R KO mouse by reconstituting T cell-deficient Rag mice with NK-1R KO T cells. These mice challanged with schistosomiasis developed abnormal liver granulomas. Granuloma size was smaller in T cell-selective NK-1R KO mice compared with granulomas in Rag reconstituted with normal T cells. Splenocytes and granuloma cells from NK-1R KO mice made less IFN-gamma. The mice also made less IgG2a. Thus T cell NK-1R is important for IFN-gamma regulation.     

A study of cytokine production in acute graft-vs-host disease

The role of cytokines in the development of acute graft-vs-host disease (GVHD) was investigated in B6AF1 mice that were injected with parental A/J lymphocytes. Splenocytes from GVH mice exhibited an increased capacity to produce interleukin (IL)-1, IL-6, and TNF-a when stimulated in culture with lipopolysaccharide (LPS). This enhanced capacity was diminished following in vivo treatment with immunosuppressive drugs. Concanavalin A-stimulated GVH spleen cells produced significantly lower levels of IL-2 but higher levels of interferon-gamma (IFN-gamma) than did syngeneic spleen cells. Immunosuppressive therapy in vivo increased the capacity of GVH spleen cells to produce IL-2. However, immunosuppressants differed in their effects on IFN-gamma production. Sch 24937 (6-bromo-5-chloro-2-[1-(methylsulfonyl)acetyl] 3-(2-pyridyl)indole) enhanced or had no effect while cyclosporin A consistently decreased the capacity of splenocytes to produce this lymphokine. These results indicate that the capacity of GVH splenocytes for cytokine production can be differentially affected by the actions of some pharmacological agents. The data also indicate that there may be differential regulation of the production of IL-2 and IFN-gamma by the Th1 subset in the GVH spleen.     

Dietary conjugated linoleic acid decreased cachexia,macrophage tumor necrosis factor-alpha production,and modifies splenocyte cytokines production

The effect of conjugated linoleic acid (CLA) on macrophage functions were studied in vitro, in vivo, and ex vivo. In RAW macrophage cell line, CLA (mixed isomers) was shown to inhibit lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha) production. Two CLA isomers, c9,t11 and t10,c12, were tested on RAW cells and it was found that the c9,t11 was the isomer responsible for the inhibition of LPS-induced TNF-alpha production. BALB/c mice were used to determine the effect of dietary CLA on body weight wasting and feed intake after LPS injection. CLA was protective against LPS-induced body weight wasting and anorexia. Plasma TNF-alpha levels after LPS injection were lower in the CLA group compared with the corn oil-fed control group 2 hr post-LPS injection. In a separate experiment, 30 mice were fed a CLA-supplemented diet or a corn oil-supplemented diet for 6 weeks and peritoneal resident macrophages were obtained for measuring TNF-alpha and nitric oxide production after in vitro exposure to interferon-gamma (IFN-gamma) and/or LPS. TNF-alpha production was not found to be different in peritoneal macrophages from mice fed the dietary treatments, but less nitric oxide was produced in macrophages from CLA-fed mice upon stimulation when compared with macrophages from control-fed mice. Splenocytes were also collected from the mice fed the dietary treatments and stimulated to produce cytokines in culture. Supernatant was used to run cytokine enzyme-linked immunoabsorbant assays. Interleukin-4 (IL-4) was decreased in CLA-fed mice when splenocytes were stimulated with concanavalin A (Con A) for 44 hr; however, IL-2 and the IL-2-to-IL-4 ratio were elevated.     

Effects of iNOS inhibitor on IFN-gamma production and apoptosis of splenocytes in genetically different strains of mice infected with Toxoplasma gondii

To evaluate the role of nitric oxide (NO) in IFN-gamma production and apoptosis of splenocytes in genetically different strains of mice with toxoplasmosis, BALB/c (a toxoplasmosis resistant strain) and C57BL/6 (a toxoplasmosis susceptible strain) mice were infected with Toxoplasma gondii cysts orally and subsequently injected intraperitoneally with aminoguanidine, an iNOS inhibitor (AG; 35 mg/kg per mouse daily for 14 days). When BALB/c or C57BL/6 mice were infected with T. gondii without AG treatment, number of brain cysts, NO and IFN-gamma production by splenocytes, and percentages of apoptotic splenocytes were increased compared to uninfected control mice without AG treatment. AG treatment increased the number of brain cysts, and reduced NO and IFN-gamma production in T. gondii-infected C57BL/6 mice. In contrast, in T. gondii-infected BABL/c mice, the number of brain cysts, and NO and IFN-gamma production of splenocytes was not altered by treatment with AG. However, the percentages of apoptotic splenocytes in T. gondii-infected BALB/c or C57BL/6 mice were not affected by AG treatment. These results suggest that NO modulates IFN-gamma production in T. gondii-infected C57BL/6 mice, and that NO is involved in mediating a protective response in toxoplasmosis susceptible, but not resistant, mice strain during acute infection.     

IFN-gamma production is specifically regulated by IL-10 in mice made tolerant with anti-CD40 ligand antibody and intact active bone

We have developed a strategy to induce tolerance to allografts, involving cotransplantation of allogeneic intact active bone and transient anti-CD40 ligand mAb therapy. Tolerance induced by this approach in C57BL/6 mice receiving BALB/c hearts is not mediated by deletional mechanisms, but by peripheral regulatory mechanisms. Tolerance is associated with diminished ex vivo IFN-gamma production that is donor specific, and a reduction in the frequency of IFN-gamma-producing cells. Splenocytes from mice tolerant to BALB/c grafts, but sensitized to third-party C3H skin grafts, demonstrated normally primed ex vivo IFN-gamma responses to C3H stimulators. Neutralizing anti-IL-10 and anti-IL-10R, but not anti-TGF-beta, anti-IL-4, or anti-CTLA-4, Abs restored the ex vivo IFN-gamma response to BALB/c stimulators. There was no significant difference in IL-2 or IL-4 production between tolerant and rejecting mice, and anti-IL-10 mAbs had no effect on IL-2 or IL-4 production. The Cincinnati cytokine capture assay was used to test whether suppression of IFN-gamma production in vivo was also a marker of tolerance. In naive mice, we observed a dramatic increase in serum IFN-gamma levels following challenge with allogeneic BALB/c splenocytes or hearts. Tolerant mice challenged with allogeneic BALB/c splenocytes or hearts made significantly less or undetectable amounts of IFN-gamma. No IL-4 or IL-10 production was detected in tolerant or rejecting mice. Collectively, our studies suggest that active suppression of IFN-gamma production by IL-10 is correlated with, and may contribute to, tolerance induced with intact active bone and anti-CD40 ligand mAbs.     

NK cells activated in vivo by bacterial DNA control the intracellular growth of Francisella tularensis LVS

We demonstrated previously that mice treated with bacterial or oligonucleotide DNA containing unmethylated CpG motifs are transiently protected against lethal parenteral challenge with the intracellular bacterium Francisella tularensis Live Vaccine Strain (LVS). Here we explore the cellular basis of this protection. Wild-type mice that were treated with CpG oligonucleotide DNA and challenged with a lethal dose of LVS survived, while mice lacking TLR9 did not. In vitro, treatment of LVS-infected macrophages and/or naive splenocytes with oligo DNA had no impact on intracellular bacterial replication. In contrast, in vitro co-culture of LVS-infected macrophages with splenocytes obtained from mice treated with oligo DNA in vivo resulted in control of intracellular LVS growth. Control was reversed by antibodies to interferon-gamma or to tumor necrosis factor-alpha and by inhibition of nitric oxide, and to a lesser degree by antibodies to Interleukin-12. Further, splenocytes from DNA-primed normal, T cell KO, B cell KO, lymphocyte-deficient scid, or perforin KO mice all controlled intra-macrophage LVS growth. Enriched DNA-primed natural killer cells, but not B cells, clearly controlled intracellular LVS growth. Thus, NK cells contribute to DNA-mediated protection by production of cytokines including IFN-gamma and TNF-alpha, resulting in nitric oxide production and control of intracellular Francisella replication.     

Production of cytokines by peritoneal macrophages and splenocytes after exposures of mice to low doses of X-rays

We have shown previously that irradiations of mice with 0.1 or 0.2 Gy of X-rays stimulate anti-tumour cytotoxic activities of peritoneal macrophages and splenocytes enriched for NK lymphocytes and suppress the development of pulmonary tumour colonies. The up-regulated cytotoxicities were related to the production of nitric oxide by macrophages, and perforin and Fas ligand by the splenocytes, but specific blockade of these pathways did not totally suppress the effector cell-mediated cytolysis of the tumour target. Hence, other factors such as cytotoxic/cytostatic cytokines might have been produced by the effector cells. To test this possibility peritoneal macrophages and splenocytes were isolated from BALB/c mice which had been either once or tentimes whole body-irradiated with the total doses of 0.1 and 0.2 Gy of X-rays and assayed for the levels of IL-1beta, IL-2, IL-12, IFN-gamma and TNF-alpha in the incubation medium using the respective ELISA kits. The results demonstrate that both single and multiple exposures to the two low doses of X-rays significantly stimulate secretion of IL-1beta, TNF-alpha and IL-12 by macrophages and IL-2 and IFN-gamma by splenocytes, but the kinetics and magnitude of the induced changes in the production of these cytokines differ between the two irradiation protocols.     

Decreasing endogenous glucocorticoids alters the ability of bone marrow cells to produce nitric oxide in response to stimulating agents

Glucocorticoids (GC) are essential for the body to maintain homeostasis. Patients with adrenal insufficiencies suffer from numerous health related problems including increased mortality due to sepsis. Here, we examine bone marrow (BM) cells from mice with adrenal insufficiency for their ability to produce nitric oxide (NO). Mice were injected with metyrapone (MR), an agent that selectively blocks glucocorticoid synthesis. BM cells were removed and tested for NO production. The stimulating agents LPS, TNF-alpha, IL-1beta, and IL-4 were all able to synergize with IFN-gamma, stimulating large concentrations of NO compared to normal mice. An important finding is that BM from injected mice produces NO in response to LPS alone, while normal BM cells do not. Experiments with anti IFN-gamma antibody demonstrate that, in MR injected mice, LPS alone stimulated sufficient quantities of IFN-gamma necessary for NO production. Our results demonstrate that reducing GCs alters regulation of NO production by BM cells at several levels.     

Estrogen regulation of nitric oxide and inducible nitric oxide synthase (iNOS) in immune cells: implications for immunity, autoimmune diseases, and apoptosis.

Nitric oxide plays a central role in the physiology and pathology of diverse tissues including the immune system. It is clear that the levels of nitric oxide must be carefully regulated to maintain homeostasis. Appropriate levels of nitric oxide derived from iNOS assist in mounting an effective defense against invading microbes. Conversely, inability to generate nitric oxide results in serious, even fatal, susceptibility to infections. Further, dysregulation or overproduction of nitric oxide has been implicated in the pathogenesis of many disorders, including atherosclerosis, neurodegenerative diseases, inflammatory autoimmune diseases, and cancer. Therefore, depending upon the levels of nitric oxide generated, the potential exists for nitric oxide to behave like a "double-edged" biological sword. Taking these issues into consideration, it is thus pivotal to understand the regulation of nitric oxide. Nitric oxide is regulated by many endogenous factors including hormones such as estrogens. While the effects of estrogen on the generation of nitric oxide in non-immune tissues are relatively well documented, the effect of estrogen on iNOS/nitric oxide in immune cells is only now becoming apparent. Our laboratory has recently shown that estrogen treatment of mice markedly upregulates the levels of iNOS mRNA, iNOS protein, and nitric oxide in activated splenocytes. This upregulation of nitric oxide is in part mediated through interferon-gamma (IFN-gamma), a pro-inflammatory cytokine that is enhanced by estrogen. These findings are important considering that estrogens are not only involved in regulation of normal immune responses, but also are implicated in many autoimmune and inflammatory diseases. To date, there are no reviews on the effects of estrogen on immune tissue-derived nitric oxide and therefore this review will address this critical gap in the literature. Given the increasing importance of immune-tissue-derived iNOS in health and disease, studies on estrogen-induced regulation of iNOS may offer a better understanding of diseases and aid in devising new therapeutic interventions.     

Interleukin 12 and antigen independently induce substance P receptor expression in T cells in murine schistosomiasis mansoni.

Substance P (SP) regulates interferon-gamma (IFN-gamma) production through interaction with the SP receptor NK1 (SPr) on T cells at sites of inflammation. Using murine schistosomiasis, we evaluated whether SPr expression was subject to immunoregulation. Splenocytes from schistosome-infected mice cultured for < or =18 h did not express SPr, as determined by quantitative polymerase chain reaction assay. However, exposure to schistosome egg antigen (SEA) for < or =4 h induced strong receptor expression. Experiments using splenocytes fractionated with antibody-coupled, paramagnetic beads showed that induction localized exclusively to T cells. Receptor protein expression was confirmed with Western blot. Interleukin 12 (IL-12) also induced strong T-cell SPr expression. Both SEA and IL-12 remained strong inducers of T-cell SPr in lymphocytes from the IL-12 (p40) and IFN-gamma R double-knockout mouse, which suggested that SEA did not require IL-12 to induce SPr and that both worked independently of IFN-gamma. Splenocytes from wild-type mice cultured with SEA and neutralizing anti-IL-12 monoclonal antibody (mAb) also showed SPr induction. However, anti-Ia mAb inhibited SEA induction of SPR: Thus, SPr is inducible on T cells. SEA induces SPr through interaction with T-cell receptor (TCR), independently of IL-12 and IFN-gamma. IL-12 induces SPr independently of TCR activation and IFN-gamma expression. SP and its receptor, which regulate IFN-gamma production, are probably part of the IL-12-Th1 circuit.     

Role of interferon regulatory factor-1 in double-stranded RNA-induced iNOS expression by mouse islets.

Environmental factors, such as viral infection, have been implicated as potential triggering events leading to the initial destruction of pancreatic beta cells during the development of autoimmune diabetes. Double-stranded RNA (dsRNA), the active component of a viral infection that stimulates antiviral responses in infected cells, has been shown in combination with interferon-gamma (IFN-gamma) to stimulate inducible nitric oxide synthase (iNOS) expression and nitric oxide production and to inhibit beta cell function. Interferon regulatory factor-1 (IRF-1), the activation of which is induced by dsRNA, viral infection, and IFN-gamma, regulates the expression of many antiviral proteins, including PKR, type I IFN, and iNOS. In this study, we show that IRF-1 is not required for dsRNA + IFN-gamma-stimulated iNOS expression and nitric oxide production by mouse islets. In contrast to islets, dsRNA + IFN-gamma fails to induce iNOS expression or nitric oxide production by macrophages isolated from IRF-1(-/-) mice; however, dsRNA + IFN-gamma induces similar levels of IL-1 release by macrophages isolated from both IRF-1(-/-) and IRF-1(+/+) mice. Importantly, we show that dsRNA- or dsRNA + IFN-gamma-stimulated IRF-1 expression by mouse islets and peritoneal macrophages is independent of PKR. These results indicate that IRF-1 is required for dsRNA + IFN-gamma-induced iNOS expression and nitric oxide production by mouse peritoneal macrophages but not by mouse islets. These findings suggest that dsRNA + IFN-gamma stimulates iNOS expression by two distinct PKR-independent mechanisms; one that is IRF-1-dependent in macrophages and another that is IRF-1-independent in islets.     

Stimulation of cytokine and nitric oxide production by acyclic nucleoside phosphonates.

Several antiviral acyclic nucleotide analogues activate expression of genes for cytokines, such as TNF-alpha, IL-10 in macrophages and IFN-gamma in splenocytes. This is an underlying mechanism for substantially enhanced production of nitric oxide generated by IFN-gamma. More lipophilic prodrugs, bis-POM-PMEA and bis-POC-PMPA, are cytocidal for macrophages and thus inhibit nitric oxide formation.     

CD1d1-dependent control of the magnitude of an acute antiviral immune response

CD1d1-restricted NK T (NKT) cells rapidly secrete both Th1 and Th2 cytokines upon activation and are therefore thought to play a regulatory role during an immune response. In this study we examined the role of CD1d1 molecules and NKT cells in regulating virus-induced cytokine production. CD1d1-deficient (CD1KO) mice, which lack NKT cells, were infected with lymphocytic choriomeningitis virus, and spontaneous cytokine release from splenocytes was measured. We found that CD1KO mice produce significantly higher amounts of IL-2, IL-4, and IFN-gamma compared with wild-type controls postinfection. Depletion studies of individual lymphocyte subpopulations suggested that CD4+ T cells are required; however, isolation of specific lymphocyte populations indicated that CD4+ T cells alone are not sufficient for the increase in cytokine production in CD1KO mice. Splenocytes from lymphocytic choriomeningitis virus-infected CD1KO mice continued to produce enhanced cytokine levels long after viral clearance and cleared viral RNA faster than wild-type mice. There was no difference in the number of splenocytes between uninfected wild-type and CD1KO mice, whereas the latter knockout mice had an increased number of splenocytes after infection. Collectively, these data provide clear evidence that the expression of CD1d1 molecules controls the magnitude of the cell-mediated immune response to an acute viral infection.     

Interleukin-12 but not interleukin-18 is required for immunity to Trypanosoma cruzi in mice

Protective immunity to the parasite Trypanosoma cruzi in mice depends on a pro-inflammatory T cell response involving the production of interferon-gamma (IFN-gamma). In conjunction with interleukin-12 (IL-12), IL-18 promotes the synthesis of IFN-gamma and a T helper type 1 immune response. We investigated the requirements of IL-12 and IL-18 in murine T. cruzi infection by use of C57BL/6 mice genetically deficient in either cytokine. IL-12p40(-/-) mice succumbed to infection at doses of 100 parasites, whereas IL-18(-/-) and wild-type mice resisted infectious doses up to 1000 parasites to the same extent. Levels of parasitemia were comparable between the latter groups, as were tissue parasite burdens according to quantitative real-time PCR. In contrast, IL-12p40(-/-) mice displayed vastly increased levels of parasites both in blood and in tissue. IFN-gamma concentrations in the serum of infected mice and in supernatants of splenocytes stimulated in vitro were decreased in IL-18(-/-) mice, whereas in IL-12p40(-/-) mice, IFN-gamma was undetectable in the serum and drastically reduced in cell supernatants. Levels of IL-12 production were generally comparable between wild-type and IL-18(-/-) mice, as were levels of IL-4, IL-2 and nitric oxide. Thus, the requirement for endogenous pro-inflammatory cytokines for a protective murine immune response against T. cruzi is satisfied by the expression of IL-12, while IL-18 is dispensable.     

The role of interleukin 6 in interferon-gamma production in thermally injured mice

Following traumatic injury, patients suffer from compromised immunity increasing their susceptibility to infection. Previous studies from this laboratory demonstrated that female BALB/c mice subjected to a 15% total body surface area (TBSA) scald injury exhibit a decrease in cell-mediated immunity 10 days post-burn. Studies described herein revealed that concanavalin A (Con A; 2 microg/ml)-stimulated splenocytes from sham treated animals produced 3557+/-853 pg/ml of IFN-gamma while splenocytes from burn injured animals released two-fold more cytokine (P<0.05). To determine whether leukocyte production of IFN-gamma was under the influence of macrophages, splenic macrophage supernatants generated from burned animals were incubated with splenic lymphocytes from sham and burn animals. The amount of IFN-gamma released by lymphocytes from sham animals increased when cultured with macrophages from burned mice (P<0.05). This suggests that the increase in IFN-gamma production by unfractionated splenocytes in burned mice relative to sham treated animals is macrophage-dependent. Macrophage supernatants from burned mice released twice as much IL-6 as supernatants from sham animals (P<0.05), and when IL-6 was blocked in vivo, the amount of IFN-gamma production in burned mice decreased to sham levels (P<0.05). Thus, IL-6 mediates IFN-gamma production following burn.     

Candida albicans double-stranded DNA can participate in the host defense against disseminated candidiasis

In the present work, we studied the in vitro immunomodulatory properties of double-stranded Candida albicans DNA and its protective effect in murine disseminated candidiasis. DNA induced the production of TNF-alpha by peritoneal macrophages and splenocytes in vitro through a chloroquine-dependent mechanism. Yeast DNA acted synergistically with IFN-gamma in triggering the secretion of nitric oxide by macrophages and enabled them to stimulate the proliferation of T cells in response to soluble anti-CD3. The effect of DNA on splenocytes is associated with an enhanced synthesis of IFN-gamma, IL-2 and IL-10. In vivo, DNA decreased the mortality and lowered the kidney contamination in mice intraperitoneally inoculated with C. albicans simultaneously with an increase in the specific proliferative response and cytokine production. The present results indicate that C. albicans DNA can provide protection against disseminated infection.     

IL-10 fails to inhibit the production of IL-18 in response to inflammatory stimuli

IL-10 is an inhibitor of the production of pro-inflammatory cytokines such as IL-12 and IL-1beta, but it is not known whether it can inhibit the production of IL-18. Therefore, a variety of in vivo and in vitro models were used to determine whether IL-10 is an inhibitor of IL-18 production. Infection of IL-10-/- mice with Toxoplasma gondii results in increased levels of IL-12 in the serum and in recall responses compared to wild type (WT) mice. Surprisingly, although infection resulted in increased levels of IL-18 in serum, there were no differences between WT and IL-10-/- mice. Moreover, splenocytes from infected WT and IL-10-/- mice produced similar levels of IL-18 and addition of exogenous IL-10 did not inhibit their production of IL-18. To address whether endogenous IL-18 inhibitors were masking increased IL-18 production in the IL-10-/- mice, expression of IL-18 binding protein was examined using RT-PCR. Although infection leads to increased expression of IL-18BP mRNA, no difference was seen between WT and IL-10-/- mice. In addition, splenocytes from IL-10-/- mice produced elevated levels of nitric oxide (NO) compared to WT mice, and NO has been shown to inhibit activity of interleukin-1 converting enzyme (ICE), which is required for IL-18 production. However, the addition of an inhibitor of NO production did not alter the levels of IL-18 produced. Finally, analysis of the levels of cytokine mRNA of macrophages stimulated with LPS and IFN-gamma revealed that although IL-10 is a potent inhibitor of IL-12 mRNA accumulation, it did not inhibit IL-1beta or IL-18. Together, these data indicate that IL-10 is not an inhibitor of the production of IL-18.     

Differential expression and tissue compartmentalization of the inflammatory response following thermal injury

Studies have shown that both animal tissue-fixed immune cells and human peripheral blood mononuclear cell (PBMC) functions are altered after burn injury. Additional studies suggest that the burn injury-induced alterations in these divergent cell populations from different species are similar. It remains unknown, however, whether the observed changes in animal tissue-fixed immune cell function following thermal injury also occurs to a similar extent in the PBMC population. The aim of our study was to compare PBMC and tissue-fixed immune cell functions from the same animal using a murine burn model. At 7 days post-burn, mice were more susceptible to sepsis and delayed type hypersensitivity responses were suppressed. Splenocytes isolated from injured mice displayed suppressed proliferation and increased IL-10 production. In contrast, PBMC from injured mice displayed suppressed proliferation, IL-2 and IFN-gamma production. Splenic macrophage nitric oxide, PGE(2), TNF-alpha, IL-6 and IL-10 production was enhanced post-burn and IL-12 production was suppressed. PBMC from such animals displayed enhanced PGE(2) production and suppressed IL-6 and IL-12 production. These results indicate that while an immunosuppressive Th(2) phenotype (increased IL-10 and/or suppressed IL-2, IFN-gamma) was induced in both the splenic and PBMC compartments post-injury, differential expression and dimorphism in the response also exists. Thus, the assessment of only PBMC function in burn patients may not accurately reflect the patient's actual immune status at the tissue level.