AN APPROACH FOR STUDIES OF MEDIATOR-INDUCED LEUKOCYTE ROPING IN THE UNDISTURBED MICROCIRCULATION OF THE RAT MESENTERY

1 Although intravital microscopy is the method of choice for observati on of inflammatory leukocyte rolling and adhesion in small venules in vivo, a problem with this technique is that surgical exposure of suita ble tissues per se triggers the rolling mechanism. In this study, we d escribe an approach to investigate induction of rolling in undisturbed microvessels. For this purpose, intravital microscopic observation of leukocyte rolling and adhesion in the rat mesentery was combined with histological determination of the intravascular concentrations of pol ymorphonuclear and mononuclear leukocytes (PMNL and MNL). 2 By relatin g the histologically determined number of intravascular leukocytes to either microvessel volume or to the erythrocyte concentration, the bas eline MNL and PMNL content was found to be 3-6 fold higher in venules than in systemic blood. This increase in microvessel leukocyte concent ration did not seem to be related to leukocyte-endothelium interaction s, because the leukocyte concentration was similarly elevated in arter ioles where rolling and adhesion did not take place. 3 Preparation of the rat mesentery for intravital microscopy time-dependently increased the venular PMNL concentration to over 100 fold the systemic PMNL con centration 45 min after exteriorization of the small intestine. The MN Ls were much less responsive to the preparative manipulation. By treat ment with the polysaccharide fucoidin (inhibits rolling but not firm a dhesion per se), or by use of intravital microscopy immediately before tissue fixation, approximately 90% of the accumulated venular PMNLs w ere found to represent rolling cells. 4 Intraperitoneal injection of 1 0(-3) M histamine increased the venular PMNL (but not the MNL) concent ration to almost 50 fold the systemic PMNL value. The histamine respon se did not vary with venular diameter, and the relative contribution o f rolling vs firmly adherent cells to the PMNL, accumulation was again approximate to 90%. Intraperitoneal injection of leukotriene C-4, but not prostaglandin E-2, caused a significant increase in venular PMNL concentration. 5 Systemic treatment with the anti-P-selectin monoclona l antibody PB1.3 had no effect on the histamine-induced venular PMNL a ccumulation (i.e. rolling) in female Wistar or male Sprague-Dawley rat s. On the other hand, identical treatment with PB1.3 very effectively inhibited the histamine-induced PMNL, response in the mesentery of rab bits. 6 In conclusion, we have shown that a histologically determined increase in leukocyte concentration in rat mesenteric venules may be u sed as an index of mediator-induced leukocyte rolling if the relative contribution of rolling and firm leukocyte adhesion is first determine d, for example by the means described in this study. This relatively s imple approach may be very useful for studying various aspects of leuk ocyte rolling when the 'spontaneous' rolling triggered by preparation of tissues for intravital microscopy is undesirable.