Granulocytes in Acute Lung Vascular Injury in Unanesthetized Sheep

To assess further whether platelet aggregation alone increased lung vascular permeability, we examined in another group of sheep the effects of ADP-induced platelet aggregation. Pulmonary lymph How increased from 7.88 ± 2.09 to 10.35 ± 2.56 ml/hr and the transvascular protein clearance increased from 6.57 ± 1.67 to 8.87 ± 2.35 ml/hr. When the microvascular pressure was increased after platelet aggregation, the increase in lymph How was associated with a decrease in lymph/ plasma protein concentration ratio. These changes could be explained solely by an increase in the pulmonary microvascular pressure indicating that ADP-induced platelet aggregation does not increase lung vascular permeability. In summary, sequestration and activation of granulocytes mediated lung vascular injury after thrombininduced pulmonary intravascular coagulation. Platelet aggregation induced by ADP infusion did not increase lung vascular permeability, and the increased permeability after thrombin was not inhibited by platelet depletion, indicating that granulocytes are the formed elements responsible for the increased permeability after thrombin-induced intravascular coagulation.

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Malik AB, van der Zee H. Thrombin-induced pulmonary insufficiency. Thrombosis Res 1977; 11:497-502 Ohkuda K, Nakahara K, Weidner WJ, Binder AS, Staub NC. Lung fluid exchange after uneven pulmonary artery obstruction in sheep. Circ Res 1978; 53:152-61 Malik AB, van der Zee H. Lung vascular permeability following progressive pulmonary microembolization . J Appl Physiol 1978; 45 :590-97 Johnson A, :\falik AB. Effect of granulocytopenia on extravascular lung fluid accumulation after microembolism. Am Rev Respir Dis 1980; 122 :561-66 Flick MR, Perel A, Staub NC. Leukocytes are required for increased lung microvascular permeability after microembolization in sheep. Circ Res 1981 ; 48:344-51

Granulocytes in Acute Lung Vascular Injury in Unanesthetized Sheep* Kenneth L. Brigham, M.D.; ]ames E. Loyd, M.D.; john H . Newman, M.D.; ]ames R. Snap,Jer, M.D.; Martin L . Ogletree, Ph .D.; and Denis K. English, Ph.D.

substances which activate granulocytes are W heninfused intravenously into unanesthetized animals, the lung circulation responds by vasoconstriction and increased permeability resulting in edema. We shall discuss responses of unanesthetized sheep to three different interventions, all thought to act primarily by •Pulmonary Circulation Center, Vanderbilt University School of Medicine, Nashville. Supported by : NIH Grant No. 19153 ( SCOR in Pulmonary Vascular Disease) Reprint requests: Dr. Brigham, Rm B1308, Pulmonary Circulation Center, Medical Center North, Vanderbilt University, Nashville 37232

56S 24TH ASPEN LUNG CONFERENCE

activating granulocytes. In each case, the two responses, that is, pulmonary vasoconstriction and increased permeability, appear to be separate, both temporally and mechanistically. GRAM-NEGATIVE ENDOTOXIN

When E coli endotoxin is infused into sheep, it causes leukopenia and stasis of granulocytes in the lung circulation. 1 The pulmonary vascular response as we have reported in the literature consists of an initial phase of marked pulmonary hypertension with increased lung lymph How and decreased lung lymph protein concentration and a later phase when pulmonary vascular pressures are stable and there is high How of proteinrich lymph indicating increased microvascular permeability. When animals are treated with arachidonate cyclooxygenase inhibitors (either indomethacin or meclofenamate), the initial pulmonary hypertension following endotoxemia does not occur, but the late phase increase in permeability occurs at least to the same extent as in untreated animals.2 In contrast, when animals are depleted of circulating granulocytes prior to infusing endotoxin, the initial pulmonary hypertensive response to endotoxemia is unaffected, but the late phase increase in permeability is much less than in animals with normal circulating granulocyte counts. 3 We conclude that the pulmonary vascular response to endotoxemia consists of initial pulmonary vasoconstriction mediated by cyclooxygenase products of arachidonic acid but not dependent upon the presence of circulating granulocytes, and a later phase of increased lung vascular permeability requiring the presence of circulating granulocytes for its full expression. CoMPLEMENT ACTIVATED AUToLoGous PLASMA

When autologous plasma, which has been incubated in vitro with zymosan to activate complement, is infused into sheep, it causes an initial phase of pulmonary hypertension with increased flow of protein-poor lymph. Several hours later, a period of normal pulmonary vascular pressure with high flow of protein-rich lymph indicates increased permeability. The response is at least qualitatively similar to the response to gram-negative endotoxemia. The magnitude of the initial pulmonary hypertension following infusion of zymosan-activated plasma is directly proportional to the total amount of zymosan-activated plasma infused. We have found that infusing approximately 100 ml of zymosan-activated plasma about doubles mean pulmonary artery pressure, whereas 400 ml causes pulmonary artery pressure to increase 6 fold. Infusion of zymosan-activated plasma also causes leukopenia, but the relationship between the degree of leukopenia and the total dose of zymosan is different from the relationship of pulmonary arterial pressure to the total dose of zymosan. In our experiments, the maximum leukopenia was about 25% of the baseline count (reflecting the total absence of granulocytes). We found that a dose of zymosan of 100 ml would re-

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duce circulating leukocyte count to about 50% of baseline, whereas 200 ml caused a maximum leukopenia and higher doses produced no further fall in circulating leukocyte count. The relationship of the late phase increase in permeability as reflected in lung lymph protein clearance to the dose of zymosan infused was similar to the relationship between the leukopenia and the dose of zymosan. Doses of 100 and 200 ml of zymosanactivated plasma caused only slight late phase increases in lung lymph protein clearance, whereas a dose of 250 ml caused a 3-fold increase in lymph protein clearance and doses of 400 ml of zymosan-activated plasma caused a similar response. Further evidence that the initial pulmonary hypertension and the later phase increase in permeability following zymosan-activated plasma infusion are mechanistically different is illustrated by the effects of repeated doses of zymosan-activated plasma given to the same animal over short periods of time. We gave 3 equal doses of zymosan-activated plasma to a sheep allowing only enough time between infusions for circulating white count to return to baseline levels. We found that in each case, zymosan-activated plasma caused the same degree of leukopenia, hut the magnitude of pulmonary hypertension caused by the substance decreased dramatically with repeated doses. Also, as with endotoxin, inhibitors of arachidonate cyclooxygenase prevented the initial pulmonary hypertension following the infusion of zymosan-activated plasma in sheep, but did not reduce the late phase increase in the flow of protein-rich lymph reflecting an increase in vascular permeability. As with endotoxin, we conclude that the pulmonary vascular response to the infusion of zymosan-activated plasma is 2-folrl. The initial pulmonary hypertension is mediated by cyclooxygenase products of arachidonic acid, hut the hter phase incrPase in lung vascular pe1meability is not. PHORBOL MYRISTATE AcETATE

Phorbol myristate acetate has been shown to he a potent stimulator of respiratory burst in granulocytes in vitro. We have fonnd some differences in its in vitro effect on sheep granulocytes and its effects on human granulocytes; phorbol myristate acetate causes no lysozyme release from sheep granulocytes, but is a potent stimulator of superoxide production. We infused 5 p.g/kg of phorbol myristate acetate into sheep. This dose of the agent caused leukopenia, with no change in circulating platelet count. As with endotoxin and zymosan-activated plasma, phorbol myristate acetate caused an initial period of marked pulmonary hypertension and increased flow of protein-poor lymph and at a later phase when pulmonary vascular pressures were at baseline levels, lung lymph flow was high and lymph protein concentration was high. This later phase indicates an increase in lung vascular permeability. We measured the effects of inhibitors of arachidonate eyclooxygcnase on tb~ responsP- to phorbol myristate

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acetate in sheep. We found that meclofenamate in sufficient doses to inhibit increases in lung lymph prostaglandin concentrations prevented the initial marked increase in pulmonary arterial pressure, but did not prevent the late phase increase in lymph protein clearance reflecting an increase in lung vascular permeability. CoNCLUSIONS

From these studies and other information available in the literature, we conclude that substances capable of activating granulocytes in vivo cause lung vascular injury. The lung vascular response is 2-fold: puhnonary hypertension occurring early in the course of the response and increased lung vascular permeability occurring later. The initial marked vasoconstriction appears to result from endogenous production of cyclooxygenase products of arachidonic acid. The later phase increase in permeability does not appear to be mediated by cyclooxygenase products of arachidonic acid, but does require the presence of circulating granulocytes. Since in sheep phorbol myristate acetate causes the late phase increase in permeability, but does not stimulate lysozyme release in vitro, we infer that the late phase increase in lung vascular permeability is mediated by granulocytes probably through production of free radicals locally in the lung circulation. REFERE!'IOCES

Brigham K , Bowers R, Haynes J. Increased sheep lung vascular permeability caused by E coli endotoxin. Circ Respir 1979; 45:292 2 Ogletree M, Brigham K. Indomethacin augments endotoxin induced increased lung vascular permeability in unanesthetized sheep. Am Rev Respir Dis 1979; 119 :384 3 Heflin C, Brigham K. Granulocyte cl.epleti·m prevents increaser! lung vasct:lar perm,ahility after endotoxemia in sheep. Clin Res 1979; 27 :3Wl:\

Oxygen Radical-Induced Pulmonary Edema* A Mechanism for the Production of Noncardiogenic Pulmonary Edema by Neutrophils R. M. Tate, M.D .; D. Michael Shasby, M.D.;t K. M. VanBenthuysen, M.D.; I. F. McMurtry, M.D.; and ]. E. Repine, M.D.

noncardiogenic pulmonary edema is a major A cute clinical problem whose pathogenesis is poorly understood. 1 Many studies have demonstrated a relationship between neutrophil accumulation in the lung and permeability edema, 2 -• but whether or not neu0

Webb-Waring Lung Institute, Pulmonary Division, Cardiovascular Pulmonary Research Lahoratory and Departments of Medicine and Pediatrics, University of Colorado Health Sciences Center, Denver. tPulmonary-Allergy Division, University of Virginia School of Medicine, Charlottesville. VA. Reprint requests: Dr. Tate, 4200 East Ninth, Denver 80262

LUNG FLUIDS AND SECRETIONS 57S