Effects of heat shock, stannous chloride, and gallium nitrate on the rat inflammatory response.

Heat and a variety of other stressors cause mammalian cells and tissues to acquire cytoprotection. This transient state of altered cellular physiology is nonproliferative and antiapoptotic. In this study, male Wistar rats were stress conditioned with either stannous chloride or gallium nitrate, which have immunosuppressive effects in vivo and in vitro, or heat shock, the most intensively studied inducer of cytoprotection. The early stages of inflammation in response to topical suffusion of mesentery tissue with formyl-methionyl-leucyl-phenylalanine (FMLP) were monitored using intravital microscopy. Microvascular hemodynamics (venular diameter, red blood cell velocity [Vrbc], white blood cell [WBC] flux, and leukocyte-endothelial adhesion [LEA]) were used as indicators of inflammation, and tissue levels of inducible Hsp70, determined using immunoblot assays, provided a marker of cytoprotection. None of the experimental treatments blocked decreases in WBC flux during FMLP suffusion, an indicator of increased low-affinity interactions between leukocytes and vascular endothelium known as rolling adhesion. During FMLP suffusion LEA, an indicator of firm attachment between leukocytes and vascular endothelial cells increased in placebo and gallium nitrate-treated animals but not in heat- and stannous chloride-treated animals, an anti-inflammatory effect. Hsp70 was not detected in aortic tissue from placebo and gallium nitrate-treated animals, indicating that Hsp70-dependent cytoprotection was not present. In contrast, Hsp70 was detected in aortic tissues from heat- and stannous chloride-treated animals, indicating that these tissues were in a cytoprotected state that was also an anti-inflammatory state.

Tissue-level cytoprotection.

In vitro and ex vivo tissue models provide a useful level of biological organization for cytoprotection studies positioned between cultured cells and intact animals. We have used 2 such models, primary tissue cultures of winter flounder renal secretory epithelium and ex vivo preparations of rat intestinal tissues, the latter to access the microcirculation of exposed mesentery tissues. Herein we discuss studies indicating that differentiated functions are altered in thermotolerant or cytoprotected tissues. These functions include transepithelial transport in renal epithelium and attachment and transmigration of leukocytes across vascular endothelium in response to mediators of inflammation. Evidence pointing to inflammation as a major venue for the heat shock response in vertebrates continues to mount. One such venue is wound healing. Heat shock proteins are induced early in wound responses, and some are released into the extracellular wound fluid where they appear to function as proinflammatory cytokines. However, within responding cells in the wound, heat shock proteins contribute to the acquisition of a state of cytoprotection that protects cells from the hostile environment of the wound, an environment created to destroy pathogens and essentially sterilize the wound. We propose that the cytoprotected state is an anti-inflammatory state that contributes to limiting the inflammatory response; that is, it serves as a brake on inflammation.

Surgical stress and the heat shock response: in vivo models of stress conditioning.

All forms of surgical therapy are stressful and injurious. The majority of surgical procedures are performed electively and provide an opportunity to condition the patient before surgery to maximize outcome. We have successfully protected the spinal cord and kidneys from warm ischemia-reperfusion injury with whole-body heat shock (42.5 degrees C, 15 min, HS) and recovery (37 degrees C, 6-8 h) before acute aortic occlusion. Control rabbits experienced an 88% incidence of paralysis (7/8) after acute spinal cord ischemia, while HS-pretreated animals never became paralyzed (0/9, p < 0.001). Control pig kidneys showed partial function (4/8 survival) after 90-min warm ischemia, while HS-pretreated kidneys always functioned (8/8 survival, p < 0.04). A positive temporal association was made between the HS-associated functional protection and the enhanced expression of inducible HSP70. The induction of the heat-shock response (cellular stress response) to protect tissues from lethal acute ischemia-reperfusion injury could be employed in a wide range of medical and surgical settings.

Acute stress impairs lymphocyte function but spares pancreatic endocrine function.

In the same animal, following HS and recovery, pancreatic islet function remains intact while immunologic functions are impaired. Cellular responses to thermal stresses are complex and tissue specific.

Lactation stage-dependent changes of lymphocyte subpopulations in mammary secretions: inversion of CD4+/CD8+ T cell ratios at parturition.

PROBLEM: Determination of lactation stage-dependent changes in levels of lymphocyte subpopulations in milk. METHOD: Flow cytometric assay was used to identify and assay lymphocyte subpopulations in bovine milk at different stages of lactation. RESULTS: Lymphocyte subpopulations in mammary secretions of dairy cows change during the lactation cycle. In involuting glands (dry gland), approximately 80-90% of lymphocytes were CD2+ T cells. The proportion of CD2+ T cells, however, decreased to approximately 50% at the colostral stage an fluctuated between 50 to 60% in normal (mature) milk. Throughout the lactation stages, less than 5% were B cells as identified by the monoclonal antibodies against CD21 and MHC class II antigens. Subset analysis showed, however, that the proportion of CD5+ T cells decreased from 90% in involuting gland secretions to 75% in colostrum (peripartum stage), and to approximately 40-50% in the normal (mature) milk, CD4+ T cells constituted between 45 to 55% of lymphocytes in the dry gland secretion but decreased drastically at parturition and maintained at the level below 20% throughout normal lactation. In contrast, the proportion of CD8+ T cells in the dry gland secretion was low, between 30 to 40%, but increased steadily, in an inversely-related manner with that of CD4+ T cells, to approximately 40-50% at parturition and maintained at approximately 30-40% during the normal lactation stage thereafter. Two-color immunofluorescence study revealed further that practically all of the CD8+ cells in dry gland secretions were CD2+, and approximately 40% of them were CD5-. Throughout the lactation cycle, WC1+ gamma delta T cells comprised only 2 to 5% of lymphocytes in mammary secretions. CONCLUSIONS: T lymphocyte subpopulations change dynamically during stages of the lactation cycle. The selective migration of T lymphocyte subpopulations to and from the mammary gland, and their functional roles in the immune competence and regulation of the dam and sucklings remain to be elucidated.

Albumin improves islet isolation: specific versus nonspecific effects.

1. BSA-containing solutions improve islet yields using standard collagenase digestion techniques. 2. The BSA effect on islet isolation is independent of source and lot of collagenase. 3. The BSA effect on islet isolation is not due solely to its colloid action, as HES failed to achieve the same level of improvement seen with albumin. 4. BSA can protect islets from warm ischemic injury, and the protective action appears to be unique to albumin, as HES was not as effective.

Lactation stage-dependent changes in levels of tumor necrosis factor/cachectin in milk.

PROBLEM: Determination of lactation stage-dependent changes in levels of tumor neurosis factor (TNF) in milk. METHOD: Bioassay and immunoblocking assay were used to identify and assay tumor necrosis factor (TNF; mostly TNF alpha) in bovine milk at different stages of lactation. RESULTS: TNF alpha levels in milk started to increase steadily after the onset of drying-off (weaning/involution), peaked at 4 to 6 wk prior to parturition and precipitously decreased to undetectable levels at parturition (colostrum). Thereafter, TNF alpha reappeared and maintained midlevel concentration in the mature (normal) milk throughout the rest of the lactation cycle. Analysis of cells in mammary secretions by flow cytometry revealed that elevated TNF alpha levels coincided with an increase in macrophages in the secretion from the dry period. CONCLUSIONS: These lactation stage-dependent changes in TNF alpha levels reflect differential effects that TNF alpha have on involution and prepartum remodeling of the mammary gland of the dam and on gastrointestinal development and immunoregulatory function of the suckling.