Thermal injury is associated with increased risk of infection. Abnormalities of neutrophil (PMN) function may predispose to infection after burn trauma. PMN adhesiveness (ADH), an important early response in various inflammatory states, has not been examined in burn patients. Data from our laboratory indicates that in vitro PMN ADH is markedly reduced in burn patients within 24 hours of injury. In addition, post burn PMN demonstrate abnormal binding and release of the adhesive glycoprotein fibronectin (Fn). These data support the hypothesis that burn injury results in altered PMN adhesion related in part to qualitative or quantitative abnormalities of PMN Fn. The long-term objective of this research is to identify mechanism(s) involved in defective PMN ADH post burn and to develop means of restoring ADH to normal following burn injury.
The specific aims of this proposal are first to characterize PMN adherence and aggregation in burn patients and compare to cells of normals and second, to examine PMN Fn physiology and its relationship to ADH in thermal injury. The approach to the first aim will involve determining the reversibility and stimulus specificity of altered PMN adherence and aggregation following burn trauma. The adhesive response of burn and control PMN will be demonstrated using various stimuli. The effect of purified Fn, Fn-rich cryoprecipitate or plasma on PMN ADH will aid in the isolation of inhibitory or adhesive factors in plasma. The approach to the second aim will initially involve quantitation of plasma Fn in burn patients and control subjects by radioimmunoassay (RIA). Qualitative differences in plasma Fn between the two groups of subjects will be assessed by immunoprecipitation of plasma with affinity purified antibody to Fn. PMN adhesive dysfunction in burn patients may be secondary to altered subcellular localization of Fn or abnormalities of surface receptors for Fn. Subcellular fractions of PMN will be isolated by nitrogen cavitation and differential centrifugation and immunoreactive Fn content of fractions quantitated by RIA. Subcellar localization of Fn will be confirmed by immuno-electron microscopy of ultrathin sections of PMN. The question of altered Fn receptor physiology in burn PMN will be addressed by quantifying the binding of radiolabeled Fn to adherent or nonadherent PMN in the presence or absence of stimulus. PMN synthesis of Fn will be examined in two ways: (1) demonstration of messenger RNA (mRNA) for Fn in PMN; (2) incorporation of C14 labeled leucine into PMN Fn by pulse-chase techniques. Absence of incorporated radiolabel in Fn may indicate Fn is synthesized at an earlier stage of PMN development. Consequently, hybridization techniques to demonstrate mRNA and radiolabeled amino acid pulse experiments will be performed with the HL60 promelocytic cell line which is capable of differentiating into various myeloid elements. Potential reversal of burn associated alterations in the synthesis or release of Fn or abnormalities of surface receptors for Fn will be examined in the presence of purified Fn, cryoprecipitate or plasma. These experiments may provide insight into the mechanisms of PMN adhesive dysfunction and enhanced risk of infection following thermal injury.
