Journal of Clinical Pathology, 1979, 32, 445-449

Increased interaction of vascular endothelium andleucocytes after administration of antiplatelet serum:

role in the developing vascular defectIREN B. KOVACS' AND J. P. CAEN

From the Laboratoire d'Immuno-Hematologie, HopitalLariboisiere, 2, rue Ambroise Pare, 75475 Paris CedexJO, France

SUMMARY Antiplatelet serum (APS) induced an increase in the stickiness of white cells whichmanifests itself in the increase in number of granulocytes rolling along or sticking to the venous

endothelium. Lidocaine treatment prevented the increased stickiness of white cells and, at the same

time, the microvascular haemorrhage developing after APS. It can be assumed that increasedstickiness of white cells after APS may contribute to endothelial damage.

Administration of antiplatelet sera (APS) is the mostselective method to eliminate platelets from thecirculation for the study of thrombocytopenicconditions. Although the specificity of APS wasquestioned by the early observation that such seracross-reacted with a vascular endothelial antigen(Bedson, 1922; Elliott and Whipple, 1940), haemor-rhagic vascular defects, which developed after theinjection of APS, were attributed to severe thrombo-cytopenia (Kitchens and Weiss, 1975).

Administration of APS to rats causes a rapid andprolonged disappearance of circulating platelets.However, little consideration has previously beengiven to the transient leucocytopenia that also occursafter administration of APS and the possible role ofgranulocytes in the development of the ensuingmicrovascular lesions. During experiments on themicrocirculation of rats treated with APS, a strikingincrease in the number of marginating (rolling)leucocytes was observed along the vascular wallimmediately after the APS infusion. In the presentstudy it is shown that administration of APSsignificantly increased the adhesive behaviour ofleucocytes to venous endothelium. This transienteffect may play a significant role in the vascularendothelial damage of small blood vessels thatoccurs in rats rendered thrombocytopenic by APS.

Material and methods

Antiserum to rat platelets was produced in rabbits.

'Present address: Korvin 0. Hospital, 1071-Budapest,HungaryReceived for publication 7 June 1978

The serum used in the present study (0-2 ml/rat)regularly produced thrombocytopenia of about104 platelets per microlitre of blood. The antiserumagglutinated in vitro rat platelets but did not inter-react with rat lymphocytes and granulocytes. Venousblood samples were taken from the cut rat tail. Totalleucocyte counts were obtained by the haemocyto-meter method, and differential counts were made on200 cells from the Giemsa-stained smears taken atthe same time. Platelets were counted by phasemicroscopy (Brecher and Cronkite, 1950). Male rats(Sprague-Dawley, CFY strain, 150-180 g bodyweight) were anaesthetised with pentobarbitalsodium (60 mg/kg subcutaneously supplementedwith 5 mg/kg intramuscularly at 30-minute intervals).Mesoappendix microcirculation was prepared

according to Zweifach (1965). Microvasculaturewas observed through a long-distance focusingobjective (UM-20, Leitz) and the microscopic fieldwas enlarged electronically (up to x 6000) by atelevision camera and monitor system. The diameterof the vessels was determined by the calibrated scaleof the monitor screen. The microscope was focusedsharply on the edge of the blood vessel. Long,straight venules, the diameter ranging between 40and 55 ,tm, were selected. The number of 'rolling'and 'sticking' leucocytes was recorded. A granulo-cyte was considered to be rolling if it was seen movingslowly along the endothelium at a rate less than thatof the axial stream of the venule. A granulocyte wasconsidered to be sticking if it was firmly adherent tothe endothelial surface or if it stopped rolling formore than 30 seconds during the observation period.As the handling and the exteriorisation of the

mesentery caused an initial increase in the number of445

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adhesive granulocytes (Atherton and Born, 1972), a15-minute resting period was necessary for stableblood flow with a small number of adhesive leuco-cytes. Observations were therefore always startedafter this resting period. Two types of observationwere made:1 From the microscopic field a 500 pm length ofvessel was selected by a shield on the monitor-screen.A line perpendicular to the axis of the vessel wasdrawn on the screen in the middle of this section.Rolling cells were counted for 1 minute on bothsides of the vessel beyond this line. Sticking leuco-cytes were counted during the 1-minute observationtime along both sides of the selected vessel-length(sticking 1).2 A long venule (diameter of vessel 30-70 ,um) wasselected for observation. The microscopic field onthe screen covered an 800 pum length of the vessel.Five microscopic fields (4000 ,tm) of the straightsections of the venule were analysed by countingsticking leucocytes along both sides of the vesselwall, and these data were expressed as the total sumof sticking cells per 4000 vm vessel-length (stickingII).Under normal conditions haemorrhagic lesions

were never observed in the mesoappendix micro-vasculature. An attempt was made to quantify the

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Iren B. Kovacs and J. P. Caen

vascular defect developing regularly after 0-2 ml APSby counting haemorrhagic lesions using a low-powerobjective along the whole mesoappendix preparation2 hours after the administration of APS.

Lidocaine HCl, when employed, was administeredintravenously as a slow infusion via the tail vein bya peristaltic pump at 1 2 ml/h. The infusion wasstarted just after APS had been administered.

Results

The effect of APS on platelet, total white blood cell,and granulocyte counts is shown in Figure 1. Whilerats remained severely thrombocytopenic for theduration of the experiment (24 hours), granulocyteand leucocyte counts returned to the preinjectionlevel within 3 hours, followed by a moderateleucocytosis and granulocytosis.Soon after the administration of APS, adhesive

granulocyte counts increased significantly (Table 1).Anti-rat plasma rabbit serum or non-immune rabbitserum did not influence rolling and sticking leuco-cyte counts (results not shown).While the number of marginating cells decreased

with time, the number of sticking white cells remain-ed increased for hours. Lidocaine treatment pre-vented a drop of granulocyte counts and subsequent

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Fig. 1 Effect ofO 2ml antiplatelet serum (APS) onplatelets, total white cells, andgranulocyte counts ofperipheral blood. Mean values obtained in six rats:

platelets;- *- total WBC;- -- granulocytes.,.. I

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2 4 6 24TIME (hr) after APS

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Increased interaction of vascular endothelium and leucocytes after administration ofantiplatelet serum

Table 1 Influence ofantiplatelet serum on granulocytemargination andadherencephenomena

Minutes No. of No. ofgranulocytes (mean)afterAPS rats(0-2 ml) Rolling Sticking I

0 18 5 1-215 12 27* 6-7*30 12 53* 5-4t45 12 38* 8.2*60 11 11 11-6*120 9 13 13-4*180 7 7 85*

For the meaning of 'rolling' and 'sticking', see Methods.Difference from the values measured before administration ofAPS-0 min. *P < 0 001 tP < 0 01

elevation of white cell stickiness in response to APS(Table 2). A typical microvascular haemorrhage,seen frequently after APS, is shown in Figure 2.Lidocaine treatment prevented (P < 0-001) thesevascular defects and subsequent purpura formationin APS-injected rats.

Discussion

Thrombocytopenia produced by injection of anti-platelet serum into experimental animals has beenwidely used to study thrombocytopenic conditionsand the role of platelets in various pathologicalreactions. Other techniques used to make animalsthrombocytopenic, such as whole-body irradiationor cytotoxic drugs (Busulfan), induce concomitantsevere granulocytopenia. APS also causes reductionof white cell counts with a more severe drop ofgranulocyte counts, but these counts returned to thepreinjection level at 4-6 hours, while the plateletcount was still diminished. Platelet antiserum,therefore, allows the desired effect of producingselective thrombocytopenia without leucopenia ifthe animal is studied a few hours after the administra-tion of the serum.According to our present findings, a shift of

leucocytes (mainly granulocytes) from the circulatingto the marginated pool appears to be responsiblefor the drop in peripheral leucocyte counts afterAPS. The observed increase in the number ofmarginating and sticking leucocytes indicates anincreased endothelial leucocyte interaction.The adherence of white blood cells to the endo-

thelium of blood vessels is the first step in theleucocyte emigration process and is therefore ofgreat pathological significance (Grant, 1973).Experimental evidence indicates that in the venules,white cell endothelial adherence and emigrationper se produce endothelial damage by releasingleucocyte cytotoxic substances (Stewart et al., 1974and 1977). These foci of vessel wall damage maywell serve as a point for red cells to permeate thewalls of small vessels during severe thrombocyto-penia.

There are two possible causes of the increasedleucocyte-endothelium interaction observed afterAPS:(a) Sticking of white cells to the vessel wall has beenattributed to changes in the endothelium itself(Grant, 1973). The effect of APS can thus beexplained by direct damage to endothelial cells(McDonald and Clift, 1976), which makes themsticky for white cells.(b)Recent evidence indicates that the primarychanges may be located in or on the granulocytesthemselves (Fehr and Jacob, 1977). It has beenshown that granulocyte margination and adherenceare complement-dependent functions. APS-inducedabrupt granulocytopenia may be the result ofcomple-ment activation resulting from the aggregation andlysis of platelets (Wautier et al., 1976) after antibody/platelet interaction, or may reflect a direct APS/granulocyte interaction.

In his classical experiments, Bedson (1922) firstshowed the simultaneous need for both thrombo-cytopenia and small vessel damage in the productionof purpura. As in the case of APS, purpura occurredwithout the need for additional damage to the

Table 2 Effect oflidocaine on antiplatelet, serum-induced, increasedgranulocyte adherence and vascular defect

Minutes Controls Lidocaine-treated ratstafterAPS No. ofrats Sticking No. of Granulocytes No. ofrats Sticking No. of Granulocytes(0-2 ml) count II purpuric (X 10Omm-3) count 1I purpuric (x 1O8mm-3)

lesions lesions

0 18 4 8 215 12 23 8 6*30 12 86 0-24 8 5* 1-43*45 12 52 8 8*60 11 34 8 7*120 9 27 8-6 7 4* 0-8*

*P < 0-001 from the respective controlstO 1 mg/min for 60 min, started immediately after the administration ofAPS (total dose = 40 mg/kg/h)

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Fig. 2 Typical microvascular defect seenfrequently in the mesentery ofthe antiplatelet serum injected rat02 ml/rat/2 hours after the administration ofserum. Increased numbers ofgranulocytes sticking to the endotheliumhave always been observed at the site where haemorrhage is developing.

endothelium. APS was considered to be causingdirect vascular damage (Ackroyd, 1953) by ahypothetical cross-reaction of antiplatelet antibodieswith blood vessel endothelial antigens (Ubatubaetal., 1975).To study the role of the increased stickiness of

white cells after APS in producing endothelialdamage, lidocaine was administered to rats. Lido-caine, like other cationic local anaesthetics, has beenreported to influence cellular responses to surfacestimuli by interfering with the function of micro-tubules and microfilaments. Electron microscopicexamination of drug-treated cells has shown thatmicrotubules and microfilaments appear normal in'resting' polymorphonuclear leucocytes, but theincrease in microtubules normally observed instimulated cells was not seen after treatment(Goldstein et al., 1977). Lidocaine inhibited whitecell sticking in vivo (Giddon and Lindhe, 1972) andprevented the endothelial damaging effect of whitecells (Stewart et al., 1974). Our present finding thatlidocaine inhibited APS-induced sticking of white

cells and simultaneously prevented vascular defectswithout influencing thrombocytopenia makes ithighly probable that the two phenomena arecausally related.From the present study the following conclusions

can be reached: APS-induced increased stickinessof white cells contributes to or plays a decisive rolein the endothelial damage developing later in thiscondition. Although peripheral white cell countsare restored within a few hours, mainly by cellscoming from the bone marrow reserve pool, thevascular wall damage effect is long-lasting. For thisreason, the presence of a pathological reaction(eg, inflammation) during APS-induced severethrombocytopenia does not necessarily mean thatplatelets are not involved in this process (Giddonand Lindhe, 1972). In addition to primary haemo-static functions, platelets are assumed to be involvedas inflammatory cells in the pathogenesis of somediseases (Nachman, 1973), increasing the permeabilityof endothelium during cellular passage.As such inflammatory endothelial changes are

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Increased interaction of vascular endothelium and leucocytes after administration ofantiplatelet serum 449

found after APS with relatively few platelets in thecirculation, the importance of platelets in initiatingendothelial damage cannot be studied properly underAPS-induced thrombocytopenic conditions.

One of us (IBK) was in receipt of a post-doctoralfellowship. Support from the Fondation pour laRecherche Medicale, France, during the final stagesis gratefully acknowledged. The help of W. Nichollsin preparing the manuscript is acknowledged.

References

Ackroyd, J. F. (1953). Allergic purpura includingpurpura due to foods, drugs and infections. AmericanJournal ofMedicine, 14, 605-632.

Atherton, A., and Born, G.V.R. (1972). Quantitativeinvestigations of the adhesiveness of circulating poly-morphonuclear leucocytes to blood vessel walls. JournalofPhysiology, 222, 447-474.

Bedson, S. P. (1922). Blood-platelet antiserum, itsspecificity and role in the experimental production ofpurpura. Journal of Pathology and Bacteriology, 25,94-104.

Brecher, G., and Cronkite, E. P. (1950). Morphology andenumeration of human blood platelets. Journal ofAppliedPhysiology, 3, 365-377.

Elliott, R. H. E., and Whipple, M. A. (1940). Observationson the inter-relationship of capillary, platelet, andsplenic factors in thrombocytopenic purpura. JournalofLaboratory and Clinical Medicine, 26, 489-498.

Fehr, J., and Jacob, H. S. (1977). In vitro granulocyteadherence and in vivo margination: two associatedcomplement-dependent functions. Studies based onthe acute neutropenia of filtration leukophoresis.Journal ofExperimental Medicine, 146, 641-652.

Giddon, D. B., and Lindhe, J. (1972). In vivo quantitationof local anesthetic suppression of leukocyte adherence.American Journal ofPathology, 68, 327-338.

Goldstein, I. M., Lind, S., Hoffstein, S., and Weissmann,G. (1977). Influence of local anesthetics upon humanpolymorphonuclear leukocyte function in vitro.

Reduction of lysosomal enzyme release and superoxideanion production. Journal of Experimental Medicine,146,483494.

Grant, L. (1973). The sticking and emigration of whiteblood cells in inflammation. In The InflammatoryProcess, edited by B. W. Zweifach, L. Grant, andR. T. McCluskey, Chapter 7, pp. 205-249. 2nd ed.Academic Press, New York.

Kitchens, C. S., and Weiss, L. (1975). Ultrastructuralchanges of endothelium associated with thrombo-cytopenia. Blood, 46, 567-578.

McDonald, T. P., and Clift, R. (1976). Mechanism ofthrombocytopenia induced in mice by anti-plateletserum. Haemostasis, 5, 38-50.

Nachman, R. L. (1973). The platelet as an inflammatorycell. In Cerebral Vascular Diseases: Eighth Conference,edited by F. H. McDowell and R. W. Brennan.Grune and Stratton, New York.

Stewart, G. J., Lynch, P. R., Reichle, F. A., Ritchie,W. G. M., Smith, A., and Schaub, R. G. (1977). Theadhesion of leukocytes, erythrocytes, and noncellularmaterial to the luminal surface of natural and artificialblood vessels in vivo. Annals ofthe New York Academyof Sciences, 283, 179-207.

Stewart, G. J., Ritchie, W. G. M., and Lynch, P. R.(1974). Venous endothelial damage produced bymassive sticking and emigration of leukocytes.American Journal of Pathology, 74, 507-521.

Ubatuba, F. B., Harvey, E. A., and Ferreira, S. H. (1975).Are platelets important in inflammation? Agents and.Actions, 5, 31-34.

Wautier, J. L., Souchon, H., Cohen-Solal, L., Peltier,A. P., and Caen, J. P. (1976). C1 and human platelets_III. Role of C1 subcomponents in platelet aggregationtinduced by aggregated JgG. Immunology, 31, 595-599.

Zweifach, B. W. (1965). Current concepts of micro-circulatory behavior. Bibliotheca Anatomica, 7, 2-8.

Requests for reprints to: Professor J. P. Caen, HopitalLariboisiere, Laboratoire d'Immuno-Hematologie, 2 rueA. Pare, 75475 Paris Cedex 10, France. on M

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