Neutrophil adhesion receptor CD18 mediates remote but not localized acid aspiration injury

Neutrophil adhesion receptor CD18 mediates remote but not localized acid aspiration injury Gideon Goldman, MD, Richard Welbourn, MB, FRCS Ed, Lester Kobzik, MD, C. Robert Valeri, MD, David Shepro, PhD, and Herbert B. Hechtman, MD, Boston, Mass. Background. Acid aspiration leads to lung polymorphonuclear neutrophil (PMN) sequestration and an associated increase in permeability. Although it is known that the neutrophil adhesion receptor (CD18) plays no role in determining P M N accumulations in the region aspirated, we postulated that this P M N adhesion receptor and its endothelial ligand, intercellular adhesion molecule-1 (ICAM-7), mediate remote neutrophil sequestration. Methods. Anesthetized rabbits underwent localized aspiration of either O. I N HCl O, Iml/kg (n = 78) or saline solution (n = 18). Results. After 30 minutes leukopenia was noted, 2290 + 200 white blood cells/ram 3 (p < 0.05). At 3 hours diapedesis occurred in the aspirated segment with accumulations in bronchoalveolar lavage fluid (• of 87 + 6 P M N / m l versus control of 6 +_ 1 P M N / m l (p < 0.05). Histologic evidence of generalized lung leukosequestration occurred. The wet to dry weight ratio of the nonaspirated lung rose to 5.7 + 0.2 versus control of 3.9 + 0.7 (p < 0.05). Treatment (n = 18) with the CD 78 monoclonal antibody (*nAb) (R 75.7, 7 mg/kg) had no effect on neutrophil accumulations in the aspirated segment. However, the mAb attenuated the remote inflammatory response: early leukopenia (5790 +_ 400 white blood cells/ram3); lung leukosequestration (24 + 4 P M N / I O high-power fields) ; protein leak in bronchoalveolar lavage fluid (570 +_ 50 #g/ml) ; and edema, wet to dry weight ratio (4.9 +_ O. 1) (all p < 0.05). Treatment with the ICAM- 7 mAb (RR 1//1, 7 mg/kg) (n = 9) did not reduce neutrophil accumulations in the aspirated segment but limited the remote inflammatory response. Conclusions. Acid aspiration leads to neutrophil adhesion and edema in regions remote from those aspirated via neutrophil CD 18 and endothelial ICAM- 1. (SURGERY 7995;777:83-9.) From the Departments of Surgery and Pathology, Brigham and Women's Hospital and The Harvard Medical School; The Naval Blood Research Laboratory and The Biological Science Center, Boston University; Boston, Mass.

ACID ASPIRATION IS A COMMON and potentially lethal complication of surgery, trauma, and labor. T h e injury is characterized by neutrophil infiltration into lung tissue associated with increased p e r m e a b i l i t y ) Neutrophils are activated after aspiration to produce H202, leukotriene B4, and thromboxane Az and to become more adhesive to the vascular endothelium. 2 Prevention Supported by The National Institutes of Health grants no. GM 24891-11, GM 35141-03, HL 16714-13; the U. S. Navy Office of Naval Research contract no. N00014-88-C-0118; the Brigham Surgical Group, Inc., and The Trauma Research Foundation. This work was supported by the U. S. Navy (Officeof Naval Research contract no. N00014-88-C-0018, with the funds provided by the Naval Medical Research and Development Command). The opinions or assertions contained herein are those of the authors and are not to be construed as official or reflecting the views of the Navy Department or Naval Service at large. Accepted for publication July 1994. Reprint requests: Herbert B. Hechtman, MD, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. 11/56/59147

of neutrophil activation or rendering rats neutropenic has been shown to minimize aspiration i n j u r y ) In other experimental settings of adult respiratory distress syndrome, neutrophils have also been shown to mediate dysfunction of the microvascular barrier. T h u s neutrophil depletion before remote ischemia or sepsis reduced the subsequent lung injury. 4' 5 Recently it has been shown that the neutrophil surface glycoprotein complex, the C D 18 integrin, regulates polymorphonuclear neutrophil ( P M N ) - e n d o t h e l i a l interaction, a requisite step in the induction of permeability and edema. This neutrophil adhesion receptor consists of three a subunits: C D l l a , C D l l b , and C D l l c linked to a common/3 subunit, CD18. Inflammatory mediators such as leukotriene B4 or the complement fragment C5a up-regulate a n d / o r increase activity of C D 1 8 leading to increased adhesion. 6 In conjunction with neutrophil adhesion receptors, cytokines such as tumor necrosis factor ( T N F ) and interleukin-1 induce endothelial expression of adhesion proteins. T h u s the intercellular adhesion molecule-1 ( I C A M - 1 ) serves as SURGERY

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a ligand for CD18. 7 Up-regulation of ICAM-1 takes 2 to 3 hours and is protein synthesis dependent. Previous studies support the likelihood that after aspiration, TNF-induced endothelial activation is largely responsible for the PMN-endothelial interaction and subsequent microvascular injury, s The role of CD18 in determining the local effect of acid aspiration has recently been investigated. It has been shown that HCl-induced localized P M N accumulations were not mediated by CD18. 9 Thus anti-CD18 monoclonal antibody (mAb) did not alter neutrophil sequestration in the aspirated segment. However, we found that localized acid aspiration provokes not only a local but also a generalized pulmonary inflammatory response. 2 The purpose of this study is to examine whether the CD18 adhesion receptor mechanism regulates this remote P M N endothelial interaction after localized acid aspiration.

MATERIAL AND METHODS Animal preparation. Sixty-nine New Zealand white male rabbits weighing approximately 3 kg were used. Anesthesia was achieved with intramuscular ketamine 35 mg/kg and intravenous xylazine 5 mg/kg and maintained with xylazin e 2 mg/kg every 30 minutes. Saline solution 0.3 m l / k g / h r was infused via a carotid arterial cannula introduced aseptically via a small neck incision on the day of the experiment. All animals were placed in a supine position on 37 ~ C heating pads. A tracheostomy was performed with a 7 mm tube. Through this tube a fine polyethylene cannula, internal diameter 0.58 mm and external diameter 0.96 mm (Delmed, Inc., Canton, Mass.), was introduced, and when allowed to follow its normal trajectory, it entered the bronchus of the middle lobe of the right lung, which represented approximately one third of that lung. Evans Blue dye 0.2 mg was added to the lavage solution for later confirmation of the location of the cannula.

Preparation of solutions Hydrochloric acid. One ml 33% HC1 (Baxter Scientific Products, McGaw Park, Ill.) was mixed with 9 ml 0.9% NaC1. The final concentration was 0.1N in 0.1 ml/kg. R15.7. This purified mAb (provided by Dr. R. Rothlein, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Conn.) recognizes the CD18 glycoprotein on rabbit neutrophils. The mAb 1 mg/kg was given 2 minutes after aspiration as a single bolus. In two sham rabbits R15.7 was given to study its effect on the circulating white blood cell (WBC) count. RR1/1. This mAb (provided by Dr. R. Rothlein) recognizes ICAM-1. A purified stock solution of 10 mg/ml was diluted with saline solution to a final con-

Surgery January 7995 centration of 3 mg/ml and was infused in a dose of 1 mg/kg 2 minutes after acid aspiration. White cell count. Circulating WBCs were quantitated by phase microscopy. Experimental protocol. Four groups were Studied. Two were treated with intravenous saline solution followed by aspiration of either 0.1 ml/kg 0.1N HC1 (n = 18) or 0.1 ml/kg of 0.9% saline solution (n = 18). The other two experimental groups were treated with intravenous R15.7 l m g / k g (n = 18) or RR1/1 1mg/kg (n = 9). The intrabronchial cannula was taken out of the middle lobe after aspiration. Three hours later the animal was killed with an overdose of ketamine. A thoracotomy was performed, and the left and right lung bronchi were clamped in turn. Bronchoalveolar lavage (BAL) of the right lung, including the aspirated segment, and the left lung were then performed in sequence with the tracheostomy tube. For each lung 5 ml saline solution was lavaged three times. The combined lavage return of about 10 ml from each lung was introduced into tubes containing 0.3 ml 0.07 ethylenediamine tetraacetic acid. This BAL fluid was centrifuged at 1500 g for 20 minutes (GLC-1 centrifuge; Sorvall, Newtown, Conn.), frozen at - 2 0 ~ C, and subsequently used for assay of protein concentration with the spectrophotometric protein dye method] ~ The BAL pellet was suspended in 1 ml saline solution, and P M N s were counted after Diff-Quik staining (AHS del Caride, Inc., Aguada, Puerto Rico) to identify macrophages. The results are expressed as mean _+ SE • 10 4 P M N / m l . We have found that P M N accumulation in BAL occurs only in the aspirated segment. Therefore the remainder of the lung was not assayed for P M N diapedesis. Six rabbits from each experimental group were used for lavage. Another six were used to calculate the wet to dry weight ratio (W/d) of the nonaspirated lungs. This was done after weighing the nonaspirated, freshly harvested lung tissue, heating the segment at 90 ~ C in a gravity convention oven (Precision Scientific Group, Chicago, Ill.) for 72 hours and weighing the residuum. Six rabbits from each group were used for lung histologic examination. After death the lungs were perfused with 10% formaldehyde and then inflated with the same material to a pressure of 25 cm H20. After fixation, sections were taken from the blue dyed middle lobe and the left inferior lower lobe and were stained with hematoxylin-eosin for light microscopic analysis. All microscopic sections were interpreted in a blind fashion by a pulmonary pathologist (L.K.). Lung sequestration of P M N s was quantitated by counting alveolar septal wall PMNs. Only peripheral lung parenchyma was examined. M i croscopic fields containing other structures such as airway, large vessels, and pleura were excluded. Leukocyte entrapment was expressed as the mean number of P M N per 10 high-power fields (Xl000). Animals in which

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Fig. 1. Localized acid aspiration induces circulating leukopenia after 30 minutes. This event was prevented with CD18 mAb. * and t indicate p < 0.05 relative to control and HCl-saline groups, respectively. Evans Blue dye appeared in the nonaspirated lung were excluded. For animals treated with RR1/1, three rabbits were used for each end point. Finally, other rabbits (n = 6) were used to calculate a correlation between neutrophil sequestration and BAL protein concentration after acid aspiration. Results are presented as mean _+ SEM in text and figures. Significance between means was tested by a one-way analysis of variance and if meaningful differences were noted a nonpaired Student's t test. Significance was accepted if p < 0.05. Animals in this study were maintained in accordance with the guidelines of the Committee of Animals of the Harvard Medical School and those prepared by the Committee on Care and use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (Department of Health, Education and Welfare publication no. 78-23 [National Institutes of Health], revised, 1978). RESULTS

Thirty minutes after acid aspiration leukopenia was noted, 2290 _+ 200 W B C / m m 3, (control, 5580 + 420 W B C / m m 3) (p < 0.05, Fig. 1). At 3 hours HC1 induced neutrophil accumulations in BAL fluid of the aspiratedlobe, 88 + 8 P M N / m l (control, 6 _+ 1 P M N / ml) (p < 0.05, Fig. 2). At this time aspiration also induced pulmonary neutrophil sequestration of 75 _+ 7 P M N / 1 0 high-power fields (HPF) in the aspirated segment and 61 + 3 P M N / 1 0 H P F in the nonaspirated lung (control, 17 _+ 6 P M N / 1 0 H P F and 14 + 4 P M N / 1 0 HPF, respectively) (p < 0.05, Fig. 3). Three

hours after aspiration when neutrophils were sequestered in the lungs, there was an increase in protein concentration in BAL to 1240 + 90 #g/ml in the nonaspirated side, higher than the value after saline aspiration of 280 _+ 20 gg/ml in the nonaspirated segment (p < 0.05, Fig. 4). A correlation was found between neutrophil sequestration and protein concentration in the nonaspirated lung (Fig. 5). There was also an increase to 5.7 + 0.2 in W / d ratios (control, 3.9 _+ 0.1 in the nonaspirated side) (p < 0.05, Fig. 6). Treatment of animals with the anti-CD18 mAb R15.7 did not alter P M N sequestration in the aspirated lobe. There was an accumulation of P M N in BAL, 90 + 8 PMN/ml, and by histologic examination in the microcirculation, 70 _+ 5 P M N / 1 0 HPF. However, the remote inflammatory response was attenuated by R15.7. Circulating leukopenia was prevented, 5790 _+ 400 WBC/mm3; leukosequestration in the nonaspirated lung was reduced, 24 +_ 4 P M N / 1 0 HPF; protein leakage in BAL was also limited, 570 + 50 #g/ml, as well as the increase in the nonaspirated lung W/d, 4.9 _+ 0.1 (all, p < 0.05). Treatment of rabbits with the anti-ICAM-1 mAb RR1/1 also did not reduce P M N accumulations in the aspirated lobe but otherwise was as effective as the R15.7 in limiting the remote pulmonary inflammatory response (Figs. 2, 3, 4, and 6). DISCUSSION

The data of this study suggest that localized acid aspiration induces generalized pulmonary edema and that this event is mediated by adherent, activated neutrophils. Second, PMN-endothelial interactions, remote to

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ASPIRATED LUNG 100

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Fig. 2. Localized acid aspiration induces localized alveolar neutrophil accumulations as measured in BAL fluid of aspirated lung. Anti-CDl 8 mAb R15.7 and anti-ICAM-1 mAb RR1/1 have no effect on this event. * indicates p < 0.05 relative to control.

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Fig. 3. Localized acid aspiration induces generalized neutrophil sequestration. The mAb against neutrophil (R15.7) and endothelial (RR1/1 ) adhesion receptors moderated this event only in nonaspirated lung. * and ~-indicate p < 0.05 relative to control and HCl-saline groups, respectively.

but not at the site of aspiration, are regulated largely by C D 1 8 and I C A M - 1 adhesion receptors. Acid aspiration induces a local and remote inflammatory response. Neutrophil accumulations in the aspirated region are not dependent on C D 1 8 or I C A M - 1 . T h u s neither m A b to C D 1 8 nor to I C A M - 1

altered P M N accumulations in B A L fluid or neutrophil sequestration in the microcirculation of the aspirated segment. Other studies have reported similar results where m A b 60,3 directed against C D 1 8 did not affect H C 1 -induced local P M N accumulations in the rabbits. 9 W e believe that the local inflammatory sequence is de-

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Fig. 5. A correlation (r = 0.89, p < 0.05) was found between neutrophil sequestration and protein accumulation in nonaspirated lung. termined by lung generation of a chemoattractant that induces non-CD18-dependent diapedesis. Peptidoleukotrienes, for example, may lead to a non-CD18dependent PMN-endothelial interaction. However, the slow timing of leukosequestration over 2 to 3 hours is at variance with the rapid effect of leukotrienes over the course of 10 to 15 minutes, 11,12 making other mechanisms more likely. The endothelial leukocyte adhesion molecule-i, known to induce non-CD18-dependent adhesion, is a

potential mediator, v3 Pulmonary macrophages or mast cells activated by acid may synthesize cytokines leading to expression of endothelial leukocyte adhesion molecule-l.14 In support of this speculation, T N F antiserum reduced P M N accumulation in the aspirated segment. 8 Furthermore, we also demonstrated the ability of a protein synthesis inhibitor to limit P M N sequestration in the aspirated segment. These observations suggest endothelial synthesis of adhesion proteins. However, the precise P M N and endothelial adhesion receptors that

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Surgery January 1995 NON-ASPIRATED LUNG

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Fig. 6. Localized aspiration induces increase in W/d of nonaspirated lung. Reduction of adhesion by using mAb against PMN and endothelial adhesion receptors limited this event. * and t indicate p < 0.05 relative to control and between groups, respectively. mediate the local inflammatory event remain to be defined. To provoke a remote inflammatory response one would expect to find circulating agents that would lead to neutrophil activation. Evidence of circulating activators was found 30 minutes after aspiration when leukopenia was observed (Fig. 1). This event suggests that neutrophils were stimulated to express adhesion receptors.1 s The ability of the anti-CD 18 mAb to prevent this event supports this thesis. Studies in aspirated rats and dogs have also shown that at the time of neutropenia, platelets were synthesizing thromboxane A2 and circulating P M N started producing H202 and then thromboxane A2. We believe that this platelet derived thromboxane mediates early P M N activation because inhibition of thromboxane synthesis or thromboxane receptors eliminated the neutropenia and prevented the acid-induced P M N oxidative burst and limited remote lung leukosequestration, Studies in vitro support the likelihood that thromboxane induces neutrophil activation. Thus P M N treated with a thromboxane mimics increased oxidative activity and adhesion to cultured pulmonary microvessels. 16, 17 The adhesion was prevented with CD18 mAb. Data of this study show that after acid aspiration circulating neutrophils become activated, possibly up-regulating CD18, and are sequestered in the microvasculature of the nonaspirated lung (Fig. 3). Another study of the kinetics of P M N sequestration after acid aspiration in rats showed that at the time of neutropenia P M N started becoming entrapped in the nonaspirated lung. 3

This temporal relationship between neutropenia and sequestration together with the ability of the anti-C D 18 mAb to prevent both events indicates a causal connection. The effectivenessof the anti- ICAM - 1 mAb RR 1/1, the endothelial ligand of CD18, in reducing the delayed P M N adhesion strengthens the thesis that remote neutrophil sequestration is a result of endothelial activation and up-regulation of ICAM-1. The data, however, are not conclusive. It is possible that RR1/1 is blocking basal expression of ICAM-1 to which P M N CD18 is binding. This could explain the early leukosequestration, although the progressive neutrophil accumulations that occur during the 3-hour period of monitoring after aspiration are most consistent with newly formed ICAM-1. Adherent neutrophils seem to be responsible in large part for the microvascular barrier dysfunction after aspiration. Thus a significant correlation was found between P M N sequestration and protein concentration in BAL of the nonaspirated lung (Fig. 5). In addition, rendering rats neutropenic before aspiration prevented the increase in microvascular permeability. 3 Taken together, the data are consistent with P M N involvement and confirm our previous thesis that the process of P M N activation and adhesion to the vascular endothelium are requisites for subsequent protein leak and edema. Reduction of neutrophil sequestration with the anti-CD 18 or anti-ICAM-1 mAb was associated with reduction of the protein leak and lung weight gain. It is believed that the process of CD18-ICAM-1 interaction stimulates the P M N to release reactive oxygen species and elastase,

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leading to disruption of b a r r i e r t i g h t n e s s and increased permeability.iS, 19 In other models of adult respiratory distress s y n d r o m e n e u t r o p h i l adhesion to the mierovasculature has been found to be a key step in d e t e r m i n i n g the vascular leak. T h u s either a n t i - C D 1 8 or antiI C A M - 1 m A b limited the i n f l a m m a t o r y response after lung ischemia or after phorbol e s t e r - i n d u c e d l u n g injury.20, 21 T h e inability of C D 1 8 m A b to fully prevent neutrophil adhesion in nonaspirated segments (Fig. 3) is not due to insufficient dosage because use of 2 m g / k g (n = 2) did not further inhibit adhesion (28 _+ 6 P M N / 1 0 H P F ) . A n o n - C D 1 8 P M N c o m p o n e n t of adhesion is likely to be taking place, w h i c h is a possible e x p l a n a t i o n of the incomplete protection of m A b 15.7 in the n o n a s p i r a t e d lung. As already noted, this is true in the aspirated lobe. It is also seen in other settings. F o r instance, h e m o r r h a g i c shock or aspiration of Streptococcus pneumoniae induced P M N a c c u m u l a t i o n s in the lung that w e r e not prevented w i t h a n t i - C D 18 mAb. 9, 22 In addition, patients w i t h congenital C D 1 8 deficiency still show a c c u m u l a t i o n of n e u t r o p h i l s in the alveolar space in response to sepsis. 9 In s u m m a r y , adhesion of activated neutrophils to end o t h e l i u m is a necessary step in acid a s p i r a t i o n - i n d u c e d remote l u n g injury. N e u t r o p h i l ( C D 1 8 ) and endothelial ( I C A M - 1 ) adhesion receptors regulate the remote but not the localized i n f l a m m a t o r y response. REFERENCES

l. Utsunomiya T, Krausz MM, Dunham B, et al. Modification of inflammatory response to aspiration with ibuprofen. Am J Physiol 1982;243:H903-10. 2. Goldman G, Welbourn R, Klausner JM, et al. Localized acid aspiration leads to thromboxane dependent generalized pulmonary edema. Surg Forum 1989;40:258-60. 3. Goldman G, Welbourn R, Kobzik L, Valeri CR, Shepro D, Hechtman HB. Localized acid aspiration induces multi-system organ injury via activated neutrophils. Surg Forum 1990;41: 51-4. 4. Klausner JM, Anner H, Paterson IS, et al. Lower torso ischemia induced lung injury is leukocyte dependent. Ann Surg 1988;208:761-7. 5. Stephens KE, Ishizaka A, Wu Z, Latrick JW, Raffin TA. Granulocyte depletion prevents tumor necrosis factor mediated acute lung injury in guinea pigs. Am Rev Respir Dis 1988; 138:1300-7. 6. Tonnesen MG, Anderson DC, Springer TA, Knedler A, Avdi N, Henson PM. Adherence of neutrophils to cultured human microvascnlar endothelial cells. Stimulation by chemotactic peptides and lipid mediators and dependence upon the Mac-l, LFA-1, p150,95 glycoprotein family. J Clin Invest 1989;83:63746.

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7. Smith CW, Rothlein R, Hughes BJ, et al. Recognition of an endothelial determinant for C D 18 dependent human neutrophil adherence and transendothelial migration. J Clin Invest 1988; 82:1746-56. 8. Goldman G, Welbourn R, Kobzik L, Valeri CR, Shepro D, Hechtman HB. Tumor necrosis factor-a mediates acid aspiration induced systemic organ injury. Ann Surg 1990;212:513-20. 9. Doershcuk CM, Winn RK, Coxon HO, Harlan JM. CD 18 dependent and independent mechanisms of neutrophil emigration in the pulmonary and systemic microcirculation of rabbits. J Immunol 1990;144:2327-33. 10. Lowry OH, Rosebrough N J, Farr AL, Randall RJ. Protein measurement with the foiin phenol reagent. J Bid Chem 1951; 193:265-74. 11. McIntyre T, Zimmerman GA, Prescott SM. Leukotrienes C4 and D4 stimulate human endothelial cells to synthesize platelet activating factor and bind neutrophils. Proe Natl Acad Sci 1986;83:2204-8. 12. Bizios R, Lai LC, Cooper JA, Vecchio PJ, Malik AB. Thrombin induced adherence of neutrophil to cultured endothelial monolayers: increased endothelial adhesiveness. J Cell Physiol

1988;134:275-80. 13. Bevilacqua MP, Pober JS; Mendriek DL, Cotran RS, Gimbrone MA. Identification of an inducible endothelial leukocyte adhesion molecule. Proc Natl Acad Sci 1987;84:9238-42. 14. Klein LM, Lavker RM, Matis WL, Murphy GF. Degranulation of human mast cells induces an endothelial antigen central to leukocyte adhesion. Proc Natl Acad Sci 1989;86:8972-6. 15. Arnaout MA, Hakim RM, Todd RF, Dana N, Colten HR. Increased surface expression of an adhesion promoting glycoprotein in the granulocytopenia of hemodialysis. N Engl J Med 1985;312:457-62. 16. Goldman G, Welbourn R, Klausner JM, Paterson IS, Shepro D, Hechtman HB. Intravascular chemoattractant activate neutrophils but inhibit diapedesis [Abstract]. FASEB J 1990;4: A1253. 17. Wiles M, Welbourn R, Goldman G, Hechtman HB, Shepro D. Thromboxane induced neutrophil adhesion to aortic and pulmonary microvaseular endothelium is regulated by CD 18 [Abstract]. Fed Proc 1990;4:A908. 18. Nathan C. Neutrophil activation on biological surfaces. Massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes. J Clin Invest 1987;80:1550-60. 19. Nathan C, Srimal S, Farber C, et al. Cytokine induced respiratory burst of human neutrophils: dependence on extracellular matrix proteins and CD l l / C D 18 integrins. J Cell Biol 1989;109:1341-9. 20. Horgan M J, Wright SD, Malik AB. Protective effect of monoclonal antibody IB4 directed against leukocyte adhesion glycoprotein CD 18 in reperfusion lung injury [Abstract]. Am Rev Respir Dis 1989;139(suppl):303. 21. Barton RW, Rothlein R, Ksiazek J, Kennedy C. The effect of anti intercellular adhesion molecule-1 on phorboI ester induced rabbit lung inflammation. J Immunol 1989;143:1278-82. 22. Vedder NB, Winn RK, Rice CL, Chi EY, Arfos K-E, Harlan JM. A monoclonal antibody to the adherence promoting leukocyte glycoprotein, CD 18, reduces organ injury and improves survival from hemorrhagic shock and resuscitation in rabbits. J Clin Invest 1988;81:676-82.