Tracy A. Freeman

Renal Cell Carcinoma Induces Prostaglandin E2 and T-Helper Type 2 Cytokine Production in Peripheral Blood Mononuclear Cells

AbstractBackground: Patients with renal cell carcinoma (RCC) do not develop an effective antitumor immune response, despite significant infiltration by lymphocytes. Tumor production of immunosuppressive factors may account for this failure. The object of this study was to investigate the production of immunosuppressive mediators, especially prostaglandin E2 (PGE2), by RCC. Methods: Peripheral blood mononuclear cells (PBMC) were cocultured with conditioned medium (CM) from human RCC cell lines in the presence or absence of NS-398, a selective cyclooxygenase 2 (COX-2) inhibitor. Supernatants were analyzed for levels of PGE2, interleukin (IL)-10, IL-6, IL-2, interferon-γ, and IL-12. The effects of RCC CM on PBMC proliferation were also examined. The expression of basal and stimulated COX-2 messenger RNA in the cell lines was assessed by reverse transcriptase-polymerase chain reaction. Results: RCC CM significantly increased PGE2 production by PBMC. T-helper type 2 (Th2) cytokine production was also significantly increased. Th1 cytokines were unchanged or decreased. RCC CM increased proliferation of PBMC. Coculture with NS-398 reduced PBMC PGE2 production to below control levels and significantly decreased IL-6 production and PBMC proliferation. NS-398 had no effect on cellular production of IL-10 or Th1 cytokines. Conclusions:Human RCC inhibits the host antitumor immune response by promoting PGE2 production and Th2 cytokines in PBMC. Selective inhibition of COX-2 may have a role in abrogating this effect.

Glucocorticoid pretreatment induces cytokine overexpression and nuclear factor-κB activation in macrophages

BACKGROUND Glucocorticoids are widely used in treating inflammatory diseases. The contribution of adrenal glucocorticoids to inflammatory regulation is unknown. Endogenous glucocorticoids, as distinct from synthetic analogues, not only suppress but also enhance immune functions. Elevated circulating cortisol levels are characteristic of injured patients. In a model of trauma, an early glucocorticoid surge occurs concomitantly with decreased cellular cytokine responses. Cytokine production elevated late after injury is associated with increased mortality. We hypothesized that this glucocorticoid surge mediates the later heightened macrophage responses. MATERIALS AND METHODS The murine macrophage like cells RAW 264.7 were incubated with corticosterone (35 ng/mL), or vehicle control, for 1 h, after which the cells were washed and corticosterone-free medium added. At 0, 3, 6, 12, and 24 h after removal of the corticosterone, the cells were stimulated with lipopolysaccharide (LPS) and interferon-gamma. Supernatant tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and nitrite levels were measured. In separate experiments the effect of pretreatment with corticosterone on TNF-alpha, IL-6, and nitrite mRNA expression as well as nuclear factor-kappaB and glucocorticoid receptor activity was determined. CD14 receptor expression was determined by flow cytometry. RESULTS Glucocorticoid pretreatment caused significantly increased RAW 264.7 cell production of nitrite, IL-6 and TNF-alpha. mRNA for these inflammatory mediators was induced 6 h after the corticosterone pretreatment, and was associated with activation of nuclear factor-kappaB in the presence of activated glucocorticoid receptor. Cell surface-expression of CD14 was likewise increased. CONCLUSIONS The results of this study demonstrate a novel role for glucocorticoids and provide a mechanism for the late upregulation in macrophage function after injury.

Cyclooxygenase-2 Inhibition Improves Macrophage Function in Melanoma and Increases the Antineoplastic Activity of Interferon γ

AbstractBackground: Melanoma inhibits macrophage tumoricidal activity and increases the expression of cyclooxygenase-2 (COX-2). In this study, we sought to determine whether inhibition of COX-2 could restore macrophage function and hence maximize the antitumor activity of the immune stimulant interferon γ (IFNγ). Methods: Peritoneal macrophages were exposed to B16 melanoma-conditioned medium for 24 hours with or without the COX-2 inhibitor NS-398 and then were stimulated with lipopolysaccharide and IFNγ. Cytotoxic activity, nitrite production, and cytokine production by the stimulated macrophages were measured. In addition, B16 melanoma cells were implanted intradermally into mice treated with IFNγ (14,000 U on alternate days) alone or with a combination of IFNγ and a COX-2 inhibitor (NS-398 or nimesulide). Mice were assessed for tumor growth and survival. Results: Macrophage cytotoxicity and nitrite production were significantly suppressed by melanoma-conditioned medium (P < .01). This was prevented by 200 μM of NS-398 (P < .05). In vivo, combined treatment with IFNγ and a COX-2 inhibitor caused a significant inhibition of tumor growth (P < .01) and improved survival (P = .02) compared with controls. Conclusions: COX-2 inhibition reversed melanoma-induced suppression of macrophage function, and combined treatment of IFNγ plus a COX-2 inhibitor was maximally effective in reducing tumor growth and improving survival.

Decreased peritoneal macrophage NF-κB translocation to the nucleus in protein energy malnutrition: a role for the glucocorticoid response?

BACKGROUND & AIMS Protein energy malnutrition (PEM) induces immune suppression leading to increased morbidity and mortality. The mechanism(s) underlying PEM-mediated immune suppression remain unclear. Plasma glucocorticoid levels are elevated in PEM and it has been postulated that these increased levels may mediate macrophage (MØ) dysfunction in PEM. We have previously shown that nuclear factor kappa B (NF-kappaB) activation in response to LPS stimulation is diminished in peritoneal macrophages (PMØs) from PEM mice. We hypothesized that decreased NF-kappaB activation in lipopolysaccharide (LPS)-stimulated PMØs in PEM is mediated through increased circulating glucocorticoid levels. METHODS Mice were randomized to six groups of n = 15 each as follows: (1) control diet (24% casein) (C); (2) protein-free diet (PF); (3) mice with subcutaneously implanted corticosterone pellet on the control diet; (4) mice with subcutaneously implanted placebo pellet on the control diet; (5) adrenalectomized mice on the control diet; (6) adrenalectomized mice on the PF diet. Within each group, the mice were pair-fed for 7 days. At the end of the experimental time period, PMØs were harvested and NF-kappaB activation determined by electrophoretic mobility shift assay (EMSA). RESULTS Elevated circulating glucocorticoids diminished NF-kappaB activation but adrenalectomy failed to restore this diminution in a murine model of PEM. CONCLUSION NF-kappaB translocation to the nucleus in PEM is independent of elevated circulating glucocorticoid levels.

Altered cyclooxygenase-2 expression and nitric oxide metabolism following major elective surgery.

BACKGROUND AND AIMS Postoperative variation in immune function leads to increased susceptibility to infections. Cyclooxygenase-2 (COX-2)-generated Prostaglandin-E(2) (PGE(2)), which signals through the PGE(2) receptor (EP receptor), as well as nitric oxide metabolites (NOx), appear to be important in postoperative immune dysfunction. It is unclear, however, how these substrates and receptors change over time. This study was conducted to evaluate postoperative changes in inflammatory mediator production and monocyte COX-2 and EP receptor expression. MATERIALS AND METHODS Nineteen patients had blood drawn preoperatively and up to 1 week postoperatively. Plasma NOx levels were measured. Peripheral blood mononuclear cell (PBMC) COX-2 and EP receptor mRNA expression were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). PBMC PGE(2), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and IL-10 productions were evaluated by enzyme-linked immunosorbent assay (ELISA) kits. Statistical analyses were by ANOVA and Students t tests. RESULTS Postoperatively, PBMC mean PGE(2) and IL-6 productions were significantly increased at all time points. Mean TNF-alpha production was maximal on postoperative day 2, while mean IL-10 production was unchanged. Mean circulating NOx levels demonstrated a biphasic response decreasing early postoperatively and normalizing at postoperative day (POD) 7. PBMC COX-2 enzyme and EP receptor mRNA expression were unchanged. CONCLUSIONS Altered PBMC PGE(2) production and plasma NOx levels support a role for altered macrophage activity, which may contribute to immune dysfunction in the postoperative period.

Serum leptin levels in acute protein deprivation.

BACKGROUND Protein energy malnutrition (PEM) induces a host neuroendocrine response, reflected by significant elevations in circulating glucocorticoid levels and associated with metabolic and immune dysfunction. Leptin regulates food intake and body mass and has a significant impact on the hypothalamic-pituitary-adrenal axis (HPA). We hypothesized that leptin may be altered by and may play an important role in regulating the effects of PEM. METHODS Female Balb/c mice were used. In experiment 1, mice were pair-fed either a protein-free (0% casein) or control (24% casein) diet for 7 days. In experiment 2, mice were implanted with either a placebo or corticosterone-releasing pellet and fed the control diet for 7 days. In experiment 3, adrenalectomized mice were pair-fed either the protein-free or control diet for 7 days. Serum corticosterone and leptin levels were measured in all experiments. RESULTS PEM caused significant reductions in food intake, body weight, and total body fat, but not lean body mass. Serum corticosterone and leptin levels were significantly greater in mice fed the protein-free diet. Subcutaneous implantation of a corticosterone pellet in mice fed the control diet resulted in a significantly elevated serum leptin level compared with placebo-implanted controls. Bilateral adrenalectomy partially blunted the increased serum leptin in PEM. CONCLUSIONS Leptin may be an important mediator of weight loss and decreased food intake in PEM. Elevated serum leptin in PEM may be secondary to elevated serum corticosterone, with other factors inherent in the host response to protein restriction also contributing to elevated serum leptin.