To examine the hypothesis that surface Pselectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin xllb183), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (Pselectin-positive, PKH2-labeled), 95 + 1% (mean + SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 ± 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKIH2-labeled, P-selectinnegative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.P-selectin, a member of the selectin family which includes Eand L-selectin, is a cell-adhesion molecule of activated platelets and endothelial cells (1, 2). P-selectin (also known as CD62P, GMP-140, and PADGEM protein) is a component of the a granule membrane of resting platelets that is only expressed on the platelet surface membrane during and after platelet degranulation and secretion (1,2). A soluble form of P-selectin circulates in plasma (3).Platelet surface P-selectin mediates the adherence of degranulated platelets to leukocytes in vitro (4, 5) and in vivo (6). It has therefore been postulated that surface P-selectin-positive (degranulated) platelets may be rapidly cleared from the circulation by leukocytes (2,4,5,7,8). In apparent contradiction to this postulate, other investigators have reported that degranulated platelets are no more rapidly cleared from the circulation than control platelets (9, 10). Methods have not previously been available to directly address this issue.In this study, we used novel three-color whole blood flow cytometric methods for tracking of circulating degran...

In addition to activating T and B lymphocytes, interleukin 1 (IL-1) induces several hematologic and metabolic changes typical of host responses to infection and injury. We now report a new biological property, namely, the induction of hypotension. Rabbits given a single intravenous injection of recombinant human IL-I-beta (5 isg/kg) rapidly developed decreased systemic arterial pressure, which reached the lowest levels after 50-60 min and slowly returned to pre-IL-I values after 3 h. Associated with the hypotension, systemic vascular resistance and central venous pressure fell, while cardiac output and heart rate increased. These responses were prevented by ibuprofen given 15 min before the IL-i. A bolus injection of IL-I followed by a 2-h infusion sustained the hypotension and was associated with leukopenia and thrombocytopenia. Ibuprofen given at the mid-point of the infusion reversed the changes in all hemodynamic parameters, but had no effect on the leukopenia or thrombocytopenia. Tumor necrosis factor (TNF) also induced a shock-like state in rabbits. When the dose of IL-1 or TNF was reduced to 1 ,ug/kg, no hemodynamic changes were observed; however, the combination of these low doses of both cytokines resulted in a profound shock-like state including histological evidence of severe pulmonary edema and hemorrhage. Pretreatment with ibuprofen prevented the hemodynamic, leukocyte, and platelet changes induced by the low-dose cytokine combination, and ameliorated the pulmonary tissue damage.These results demonstrate that IL-1, like TNF, possesses the ability to induce hemodynamic and hematological changes typical of septic shock, and that the combination of IL-I and TNF is more potent than either agent alone. These effects seem to require cyclooxygenase products, and suggest that intravenous cyclooxygenase inhibitors may be of therapeutic value in patients with IL-i/TNF-mediated shock.

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IntroductionTumor necrosis factor (TNF) and IL-I are thought to mediate many of the pathophysiologic changes of endotoxemia and Gram-negative bacteremia. In these studies, heat-killed Staphylococcus epidermidis were infused into rabbits to determine whether an endotoxin (LPS)-free microorganism also elicits cytokinemia and the physiologic abnormalities seen in Gramnegative bacteremia. S. epidermidis induced complement activation, circulating TNF and IL-1, and hypotension to the same degree as did one-twentieth the number of heat-killed Escherichia coli. Circulating IL-1ft levels had a greater correlation coefficient (r = 0.81, P < 0.001) with the degree of hypotension than TNF levels (r = 0.48, P < 0.02). Leukopenia, thrombocytopenia, diffuse pulmonary capillary aggregation of neutrophils, and hepatic necrosis with neutrophil infiltration were observed to the same extent after either S. epidermidis or E. coli infusion. However, S. epidermidis infusion did not induce significant (< 60 pg/ml) endotoxemia, whereas E. coli infusion resulted in high (11,000 pg/ml) serum endotoxin levels. S. epidermidis, E. coli, LPS, or S. epidermidis-derived lipoteichoic acid (LTA) induced TNF and IL-1 from blood mononuclear cells in vitro. E. coli organisms and LPS were at least 100-fold more potent than S. epidermidis or LTA. Thus, a shock-like state with similar levels of complement activation as well as circulating levels of IL-1 and TNF were observed following either S. epidermidis or E. coli. These data provide further evidence that host factors such as IL-1 and TNF are common mediators ofthe septic shock syndrome regardless ofthe organism. (J. Clin. Invest. 1991. 87:1925-1935

Stress activates the hypothalamic-pituitary-adrenal axis through release of corticotropin releasing hormone (CRH), leading to production of glucocorticoids that down-regulate immune responses. Acute stress, however, also has proinflammatory effects that seem to be mediated through the activation of mast cells. Stress and mast cells have been implicated in the pathophysiology of various inflammatory conditions, including some in the central nervous system, such as multiple sclerosis in which disruption of the blood-brain barrier (BBB) precedes clinical symptoms. We previously showed that acute restraint stress increases rat BBB permeability to intravenous 99 Tc gluceptate and that administration of the "mast cell stabilizer" disodium cromoglycate (cromolyn) inhibits this effect. In this study, we show that the CRH-receptor antagonist Antalarmin blocks stress-induced 99 Tc extravasation, whereas site-specific injection of CRH in the paraventricular nucleus (PVN) of the hypothalamus mimics acute stress. This latter effect is blocked by pretreatment of the PVN with cromolyn; moreover, restraint stress cannot disrupt the BBB in the diencephalon and cerebellum of W/W v mast cell-deficient mice. These results demonstrate that CRH and mast cells are involved in regulating BBB permeability and, possibly, brain inflammatory disorders exacerbated by acute stress.