Nadeem Fazal

Cutaneous Expression of CRH and CRH-R: Is There a “Skin Stress Response System?”

ABSTRACT: The classical neuroendocrine pathway for response to systemic stress is by hypothalamic release of corticotropin releasing hormone (CRH), subsequent activation of pituitary CRH receptors (CRH‐R), and production and release of proopiomelanocortin (POMC) derived peptides. It has been proposed that an equivalent to the hypothalamic‐pituitary‐adrenal axis functions in mammalian skin, in response to local stress (see Reference 1 ). To further define such system we used immunocytochemistry, RP‐HPLC separation, and RIA techniques, in rodent and human skin, and in cultured normal and malignant melanocytes and keratinocytes. Production of mRNA for CRH‐R1 was documented in mouse and human skin using RT‐PCR and Northern blot techniques; CRH binding sites and CRH‐R1 protein were also identified. Addition of CRH to immortalized human keratinocytes, and to rodent and human melanoma cells induced rapid, specific, and dose‐dependent increases in intracellular Ca2+. The latter were inhibited by the CRH antagonist α‐helical‐CRH(9–41) and by the depletion of extracellular calcium with EGTA. CRH production was enhanced by ultraviolet light radiation and forskolin (a stimulator for intracellular cAMP production), and inhibited by dexamethasone. Thus, evidence that skin cells, both produce CRH and express functional CRH‐R1, supports the existence of a local CRH/CRH‐R neuroendocrine pathway that may be activated within the context of a skin stress response system.

Neutrophil depletion in rats reduces burn-injury induced intestinal bacterial translocation.

Objective: To determine whether neutrophil depletion could eradicate intestinal bacterial translocation in burn‐injured rats. Design: Prospective, randomized, controlled study. Setting: University research laboratory. Subjects: Adult male Sprague‐Dawley rats. Interventions: The rats were intravenously administered a rabbit anti‐rat neutrophil antibody causing profound neutropenia and subjected to a 30% total body surface area scald burn. Measurements and Main Results: The depletion of neutrophils from the intestine was assessed via measurements of myeloperoxidase (MPO) activity in the intestinal homogenates. In addition, the presence of activated/extravasated neutrophils in intact intestines was determined via immunohistochemical localization of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase component protein p47phox. Bacterial translocation was measured using agar cultures and by determining Escherichia coli β‐galactosidase gene via polymerase chain reaction/Southern blot analyses of mesenteric lymph node and spleen, liver, lung, and blood. MPO measurements demonstrated a six‐fold increase above the control value in the intestinal tissue in rats on day 1 postburn. The presence of activated neutrophils (expression of p47phox protein) was also markedly increased in the intestines of these rats. The increased MPO activity and p47phox expression accompanied a translocation of indigenous E. coli into the mesenteric lymph node without a spread to other organs. The administration of anti‐neutrophil antibody to burn animals prevented an increase in MPO activity and bacterial translocation. Conclusion: These studies indicate that enhanced intestinal bacterial translocation caused by burn injury could be related to the increased infiltration of activated neutrophils into the intestinal tissue after burn. The release of neutrophil products such as superoxide anion may effect intestinal tissue damage leading to bacterial translocation of indigenous E. coli.

Effect of CRF and related peptides on calcium signaling in human and rodent melanoma cells

Corticotropin releasing factor (CRF) induces a rapid, within seconds, and dose‐dependent increase in the intracellular Ca2+ in both human and hamster melanoma cells. This effect is inhibited by depletion of extracellular calcium using 3 mM EGTA and is attenuated by the CRF receptor antagonist, α‐helical‐CRF(9‐41). Other peptides of the CRF superfamily, sauvagine and urocortin, also induce increases in cytoplasmic calcium concentration but at higher concentrations than CRF. We conclude that malignant melanocytes express CRF receptors, which are coupled to activation of plasma membrane calcium channels.

Role of neutrophils in burn-induced microvascular injury in the intestine.

The present study evaluated burn-induced vascular permeability alterations of rat small intestine in vivo and assessed the effect of neutrophil depletion in burn-injured rats on the altered intestinal microvascular permeability. 125I-labeled bovine serum albumin (125I-BSA) was injected intravenously, and its leakage from circulation into the intestinal tissue was determined by measuring tissue counts of 125I-BSA. Compared with sham, vascular albumin permeability increased 1.7-fold on day 1 post-burn and 3.0-fold on day 3 post-burn in ileum. In the jejunum, albumin permeability increased 1.8- and 2.5-fold on day 1 and day 3 post-burn, respectively. Intestinal tissue edema, determined as increases in tissue water contents, was noted in both intestinal segments on day 1 post-burn; no further increase in edema was found on day 3 post-burn. Neutrophil depletion before burn injury prevented the vascular leakage of albumin and edema in the ileum and jejunum on day 1 post-burn. On day 3 post-burn, the effect of prior neutrophil depletion on vascular permeability was less marked, and edema formation was not affected at all. These findings indicate that an absence of neutrophils prevents the loss of intestinal vascular barrier properties only in the initial periods after burns.

PGE2 suppresses intestinal T cell function in thermal injury : A cause of enhanced bacterial translocation

Increased gut bacterial translocation in burn and trauma patients has been demonstrated in a number of previous studies, however, the mechanism for such an increased gut bacterial translocation in injured patients remains poorly understood. Utilizing a rat model of burn injury, in the present study we examined the role of intestinal immune defense by analyzing the T cell functions. We investigated if intestinal T cells dysfunction contributes to bacterial translocation after burn injury. Also our study determined if burn-mediated alterations in intestinal T cell functions are related to enhanced release of PGE2. Finally, we examined whether or not burn-related alterations in intestinal T cell function are due to inappropriate activation of signaling molecule P59fyn, which is required for T cell activation and proliferation. The results presented here showed an increase in gut bacterial accumulation in mesenteric lymph nodes after thermal injury. This was accompanied by a decrease in the intestinal T cell proliferative responses. Furthermore, the treatments of burn-injured animals with PGE2 synthesis blocker (indomethacin or NS398) prevented both the decrease in intestinal T cell proliferation and enhanced bacterial translocation. Finally, our data suggested that the inhibition of intestinal T cell proliferation could result via PGE2-mediated down-regulation of the T cell activation-signaling molecule P59fyn. These findings support a role of T cell-mediated immune defense against bacterial translocation in burn injury.

Effects of burn with and without Escherichia coli infection in rats on intestinal vs. splenic T-cell responses.

Objective To evaluate the effect of burn injury with and without an Escherichia coli septic complication on T-cell proliferation, interleukin-2 production, and Ca2+ signaling responses in intestinal Peyer’s patch and splenic T cells. Design Prospective, randomized, sham-controlled animal study. Setting University medical center research laboratory. Subjects Adult male Sprague-Dawley rats. Interventions Rats were subjected to a 30% total body surface area, full skin thickness burn. Infection in rats was induced via intraperitoneal inoculation of E. coli, 109 colony forming units/kg, with or without a prior burn. Measurements and Main Results Rat Peyer’s patch and splenic T lymphocytes were isolated by using a nylon wool cell purification protocol. T-cell proliferation, interleukin-2 production, and Ca2+ signaling responses were measured after stimulation of cells with the mitogen, concanavalin A. T-cell proliferation was determined by measuring incorporation of 3H-thymidine into T-cell cultures. Interleukin-2 production by T-cell cultures was measured by using enzyme-linked immunosorbent assay. Intracellular T-cell Ca2+ concentration, [Ca2+]i, was measured by the use of Ca2+-specific fluorescent label, fura-2, and its fluorometric quantification. [Ca2+]i was also evaluated by the use of digital video imaging of fura-2 loaded individual T cells. T-cell proliferation and interleukin-2 production were suppressed substantially in both Peyer’s patch and splenic T cells 3 days after either the initial burn alone or burn followed by the E. coli inoculation at 24 hrs after the initial burn. There seemed to be no demonstrable additive effects of E. coli infection on the effects produced by burn injury alone. The T-cell proliferation and interleukin-2 production suppressions with burn or burn-plus-infection insults were correlated with attenuated Ca2+ signaling. E. coli infection alone suppressed T-cell proliferation in Peyer’s patch but not in splenic T cells at 2 days postbacterial inoculation;E. coli infection had no effect on Peyer’s patch or splenic T cells at 1 day postinjury. On the other hand, burn injury alone caused a substantial T-cell proliferative suppression at 2 days postburn in both Peyer’s patch and splenic cells and a significant suppression in T-cell proliferation on day 1 postburn in Peyer’s patch but not in the spleen. Conclusion An initial burn injury suppressed T-cell proliferation at a level that it would not be further affected by a subsequent infection even if the infection by itself has the potential of suppressing T-cell proliferation. An earlier onset of T-cell suppression in Peyer’s patch cells than in the spleen with burn could be attributable to an initial hypoperfusion-related intestinal mucosal tissue injury. Overall, our study supports the concept that burn injury per se can significantly suppress T-cell mediated immunity and that the intestine is an early tissue site of such suppression.

CINC blockade prevents neutrophil Ca2+ signaling upregulation and gut bacterial translocation in thermal injury

In this study, we have evaluated the role of cytokine-induced neutrophil chemoattractant (CINC), in the upregulation of neutrophil Ca(2+) signaling in neutrophils from thermally injured rats treated with anti-CINC antibody. Additionally, we have determined the effect of the treatment with CINC antibody on the accumulation of activated neutrophils in the intestinal wall, and the effect of such accumulation on gut bacterial translocation. Measurements of myeloperoxidase (MPO) activity and immunohistochemical localization of neutrophils determined neutrophil sequestration in the rat intestine. Agar culture analyses and a specific Escherichia coli beta-galactosidase gene polymerase chain reaction was carried out to detect gut indigenous bacterial invasion into intestinal wall and extraintestinal mesenteric lymph nodes (MLN). The results showed that pretreatment of rats with anti-CINC antibody attenuated the thermal injury-induced enhancement in [Ca(2+)](i) responses in neutrophils both in the basal and Formyl-Met-Leu-Phe stimulated conditions. Moreover, treatment with the CINC antibody decreased neutrophil infiltration into the gut and attenuated thermal injury-caused translocation of bacteria into the MLN.

Role of NFAT and AP-1 in PGE2-mediated T cell suppression in burn injury.

PGE2 is known to suppress T cell proliferation and IL-2 production in many inflammatory conditions. Previous studies from our laboratory have shown that such suppression of T cell proliferation in burn and sepsis could result from alteration in T cell activation signaling molecule p59fyn. In this study, we examined the role of downstream signaling molecules NFAT and AP-1 in PGE2-mediated suppression of T cell in burn injury. These studies were carried out utilizing splenic T cells from sham and burn rats 3 days after injury. The data presented in this manuscript suggest a significant suppression of IL-2 production by T cells from burn injured rats compared with the T cells from sham rats. The suppression in T cell IL-2 production was accompanied by a decrease in the activation of NFAT and AP-1 as well as a decrease in T cell p59fyn kinase activity. The treatments of burn-injured animals with PGE2 synthesis blocker indomethacin prevented both the decrease in NFAT and AP-1 binding to IL-2 sequences. In vitro incubation of control rat T cells with PGE2 suppressed the activation of NFAT and AP-1. These results suggested that the suppression of T cell IL-2 production could result from PGE2-mediated alterations in the T cell signaling molecule p59fyn and NFAT/AP-1.

Enteral nutritional supplementation prevents mesenteric lymph node T-cell suppression in burn injury

OBJECTIVE To determine the effects of an immune-enhancing diet supplemented with glutamine, arginine, fish oil, and dietary nucleotides on mesenteric lymph node T-cell functional disturbances encountered after burn injury in rats. DESIGN A prospective animal study. SETTING University medical center research laboratory. SUBJECTS Adult male Sprague-Dawley rats. INTERVENTIONS Rats received a 30%, total body surface, full-thickness burn. Burn-injury rats received the IMPACT diet supplemented with glutamine, arginine, fish oil, and nucleotides or arginine, fish oil, and nucleotides, or an isocaloric/isonitrogenous diet without supplementation with glutamine, arginine, fish oil, or nucleotides. MEASUREMENTS AND MAIN RESULTS Two days after injury, we found a significant decrease in the proliferation and interleukin-2 production by mesenteric lymph node T cells derived from rats fed on conventional chow compared with sham rats. The burn-related suppression of mesenteric lymph node T-cell proliferation and interleukin-2 production was prevented when the rats were fed on a high-protein diet rich in glutamine, arginine, fish oil, and nucleotides. We found that the immunostimulatory effects of the enriched diet are dependent on the presence of glutamine, arginine, fish oil, and nucleotides as feeding of rats on the isocaloric/isonitrogenous diet deficient in glutamine, arginine, fish oil, and nucleotides did not prevent the burn-related suppression of mesenteric lymph node T-cell dysfunction. Finally, our studies suggested that immunostimulatory effects of the diet are mediated by prostaglandin E(2) regulation of T-cell activation signaling molecule P59fyn. CONCLUSION These results suggest that a diet rich in arginine, fish oil, and nucleotides, with and without glutamine, can effectively prevent T-cell dysfunction encountered after burn injury.

Enhanced expression of neutrophil NADPH oxidase components in intestine of rats after burn injury

We have evaluated the accumulation of neutrophils in the gut and their infiltration into the intestinal extravascular spaces in rats subjected to a 25% total body surface area scald burn. The accumulation of neutrophils was assessed via measurements of myeloperoxidase (MPO) activity in the intestinal homogenates, and the immunohistochemical localization of neutrophil NADPH oxidase component proteins (p47phox and p67phox) within the intestinal extravascular spaces determined neutrophil tissue infiltration. MPO measurements demonstrated a 12- and 21-fold increase above the control value in the intestinal tissue at day 1 and day 3 post-burn, respectively, suggesting that a substantial total tissue accumulation of neutrophils occurs in the gut after burn injury. The immunohistochemical staining procedures showed both a definitive presence of the neutrophil in the intestinal extravascular spaces and an enhanced immunoreactivity in neutrophils accumulating in intestine after burn injury. There was no evidence of either the presence of neutrophils in the extravascular regions or any significant neutrophil immunoreactivity to NADPH oxidase component proteins in the intestines of sham control rats. These findings indicate that burn injury causes an enhanced migration of circulating neutrophils into the intestinal interstitial spaces and an upregulation of NADPH oxidase activity in the infiltrating neutrophils.