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CURRENT RESEARCH REVIEW Neutrophil-Endothelial

Cell Binding in Neutrophil-Mediated Tissue Injury’

JUAN CARLOS DE LA OSSA, B.A., MASSIMO MALAGO, M.D., AND BRUCE L. GEWERTZ, M.D. University

of Chicago, Chicago, Zllinois 60637-1470

Submitted

for publication

Reperfusion of an ischemic organ has harmful effects which exacerbate the hypoxic injury occurring during blood flow interruption. Although most would agree that these reperfusion phenomena are initiated by the production of reactive oxygen species, recent data have indicated that neutrophils may be the primary mediators of the actual tissue injury. Circulating neutrophils are both attracted by and produce oxygen radicals, and these cells release other potent nonoxidative toxins (proteases, cationic proteins, and collagenases). Neutrophil binding to the microvascular endothelium is the essential step for both cellular activation and the production of free radicals and associated toxins. This review will delineate the mechanisms by which neutrophils bind and become activated and assess possible clinical uses of monoclonal antibodies and other agents in inhibiting neutrophil-mediated ischemia-reperfusion injury. The discussion is divided into (1) neutrophil-dependent and (2) endothelial-dependent mechanisms of adherence.

The Neutrophil

15, 1991

CDlla/CD18, also known as lymphocyte function-associated antigen-l (LFA-l), is expressed on all white blood cells [ 11. It has been shown to mediate unstimulated neutrophil attachment to stimulated human endothelium most likely through an interaction with the intercellular adhesion molecule (ICAMreceptor) on the endothelial cell surface. A role for LFA-1 in cytokine-induced transendothelial migration of neutrophils has also been suggested [5]. CDllb/CD18 (Mac-l, the C3bi complement receptor, CR3, or MO-~) has been isolated from polymorphonuclear leukocytes (PMN), monocytes, and natural killer cells. This glycoprotein participates in a number of functions requiring adhesion, such as neutrophil aggregation, cytotoxicity, and chemotaxis [ 1,4]. Chemotactitally stimulated neutrophils are thought to bind to endothelial cells via a Mac-l-ICAMinteraction [5, 61. Mac-l may also bind to another ligand on the endothelial cell [7]. The third heterodimer of this complex, CDllc/CD18 (p150,95, or CR4), is known to be present on both granulocytes and monocytes. It is thought to participate in monocyte adhesion, although its role in PMN binding has not been established [ 11.

INTRODUCTION

NEUTROPHIL-DEPENDENT MECHANISMS

January

ADHERENCE

Activating

Integrins

Neutrophil binding is mediated by a glycoprotein complex on the surface of the cell. This entity is composed of three related heterodimers each containing a characteristic CY subunit (either CDlla, CDllb, or CDllc) which share a common p subunit (CDlS) required for surface expression. This complex is also present in hematopoietic precursor cells and other leukocytes [l-4].

* Research supported by American Heart Association Grant 88-786 (Dr. Gewertz) and NIH Grant T32 HL607665-02 (Dr. Malago). 103

Agonists

As previously mentioned, in vitro studies show that unstimulated human neutrophils adhere spontaneously to cultured endothelial cells from human endothelial veins [l, 51. Mg2+ 1s ’ required for effective neutrophil-endothelial cell interaction [2]. A number of substances have been found to stimulate neutrophil adherence and elicit inflammation. Chemotactic peptides (C5a of the complement cascade and formyl-methionyl-leucyl-phenylalanine) and lipid mediators (leukotriene B andplatelet activating factor) substantially enhance PMN adherence in a variety of settings [ 1,3,8] by enhanced expression of adhesion glycoproteins. This increase in binding 0022-4804/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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is dose-dependent and occurs rapidly (within 2 min) [9, lo]. Phorbol diesters and the calcium ionophore A23187 also act in a similar manner to activate neutrophils 17, 111. Monoclonal Agents

Antibodies

(MoAB)

and Other Therapeutic

Monoclonal antibodies against the anti-a chain of the CD11/18 complex have been used to determine the roles of specific subunits in PMN binding. Even more importantly, this research has suggested that inhibition of neutrophil binding may have clinical applicability. Anti CDlla (LFA-1) MoAb such as R3.1 (IgGl), TS1/22 (IgGl), and Lll all markedly decrease unstimulated neutrophil attachment to cytokine-stimulated endothelium [5, 71. Anti-CDllb (Mac-l) MoAb such as LM2/1 (IgGl), OKMl (IgG2,), OKMlO (IgGB,), M1/70, and 904 (IgGl) inhibit binding of stimulated neutrophils to cultured endothelial cells [5,7,12]. In an in uiuo study, MoAb 904 administered to canines subjected to myocardial ischemia reduced the size of eventual myocardial infarctions to 54% of that seen in untreated animals [12]. Anti-P chain (CD18) MoAbs inhibit PMN endothelial adherence stimulated by chemotactic peptides, lipid mediators, phorbol esters, and the calcium ionophore A231787. MoAb 60.3, a murine IgG2a which recognizes an epitope on CD18 of both peripheral and marrow leukocytes, has been shown to lessen neutrophil-mediated tissue damage in a variety of experimental settings. In vitro application of MoAb 60.3 prevents phorbolinduced neutrophil binding to human endothelial cell cultures. In addition, MoAb-treated neutrophils also inhibit the nonlytic endothelial cell detachment provoked by phorbol esters and A231787, but have no effect on hydrogen peroxide-mediated lysis or retraction [ 111. Rabbits pretreated with MoAb 60.3 demonstrated a decreased PMN adherence in chemotaxin-treated venules and diminished PMN tissue migration. As a consequence, neutrophil-induced plasma leakage and PMN accumulation in muscle and inflammatory skin lesions were markedly inhibited. This decrease in PMN adherence has been reported to last for at least 5 hr [8]. In related experiments, Price and colleagues inserted endotoxin-soaked sponges into the subcutaneous tissue of rabbits and measured PMN migration with and without MoAb 60.3. The MoAb was found to bind quickly and effectively (99.5% of maximal binding within 5 min) and to elicit a dose-dependent reduction of in viuo PMN migration when applied at the time of endotoxin treatment. Higher MoAb doses resulted in almost total inhibition of migration [ 131. Vedder and colleagues used a rabbit model of hemorrhagic shock and resuscitation to demonstrate the effects of neutrophil binding on ischemia-reperfusion injury. MoAb 60.3 was applied before the ischemic insult in one study [14] and immediately before resuscitation

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in another [ 151. Both experiments showed increased survival in treated animals, as well as a marked reduction in organ damage. This was most prominent in the liver and gastrointestinal mucosa; injury to the lungs was not significantly reduced. Interestingly, granule release and oxidant generation were not affected by MoAb 60.3 [ll, 151. Hernandez and colleagues studied isolated feline ileal segments in which arterial flow was reduced to 1520% of control; flow was restored after 1 hr of ischemia. Animals pretreated with MoAb 60.3 experienced an attenuation of reperfusion injury as measured by a decrease in post-reperfusion microvascular permeability [16]. Unfortunately, the clinical applicability of these treatments may be limited by the potential systemic effects. MoAb 60.3 produces symptoms similar to those found in leukocyte adhesion molecule deficiency, a disease where CD18 is lacking or incomplete. Patients with this disorder suffer recurrent bacterial infections and impaired wound healing [ 1, 171. In addition to impairing neutrophil function, MoAb 60.3 also binds to the CD18 molecule on other leukocytes. In culture, MoAb 60 has been shown to reduce T-cell proliferation in response to diverse stimuli if added early in the cell line. No attenuation occurs if the MoAb is added after 48 hr of incubation. MoAb 60.3 also hinders the lytic activity of both T cells and NK cells in a dose-dependent fashion [X3]. MoAb lOF12, a second antibody against the CD18 complex, has been found to block PMN adhesion by 78% [19]. A number of other treatments have been found to reduce neutrophil-mediated ischemia-reperfusion injury. Among these, reperfusion with neutropenic blood has proved particularly successful. Hernandez et al. reperfused the previously mentioned feline ileal model in animals pretreated with antineutrophil serum. They reported a decrease in microvascular permeability similar to that seen with MoAb 60.3 treatment [16]. Smith and colleagues found an attenuation of post-ischemic tissue damage and red blood cell leakage into the gut lumen in rats pretreated with neutrophil antiserum [20]. Furthermore, reperfusion of rat small intestine with leukocytefree perfluorochemicals resulted in significant amelioration of tissue injury [21]. Korthuis and colleagues [22] and Belkin and colleagues [23] reperfused ischemic skeletal muscle in rat hind limbs with neutrophil-depleted blood. Both groups reported improvements in post-ischemit injury with neutropenic treatment. In ischemic renal injury in rats, PMN depletion was found to improve post-ischemic tubular leakage in one experiment [24], but had no effect on renal damage in another [25]. Experiments on neutrophil depletion in myocardial ischemia have yielded divergent results depending on the experimental model. Romson and colleagues reported a significant reduction in the size of myocardial infarct with neutrophil depletion before ischemia-reperfusion [26]. In contrast, O’Neill et al. found no evi-

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dence of injury amelioration in dogs submitted to a 15 min coronary occlusion and reperfused with neutropenic blood [27]. In similar experiments, Jolly and colleagues performed both 90-min and 4-hr coronary artery occlusions in dogs. They found some reduction in infarct size with neutropenic reperfusate in the first interval but reported no significant reduction of damage in the second [28]. Interestingly, neutrophil depletion had no effect on the neurologic damage that results from cardiac arrest [29]. Homing Receptors PMN also express molecules related to the lymphocyte homing receptors (HERMES homing receptor, Pgp-1 in humans) which may play a role in neutrophil adherence. MoAb MEL14 against mouse lymph node homing receptors has been shown to block both neutrophi1 and monocyte interaction with endothelial cells and decrease leukocyte accumulation [2, 30, 311. ENDOTHELIAL-DEPENDENT OF ADHERENCE: ELAM-1

MECHANISMS AND

ICAM-

The endothelial cell plays a vital role in neutrophil binding through a series of adhesion molecules expressed on its surface. At present, two endothelium adhesion molecules for neutrophil binding have been well characterized: the endothelial-leukocyte adhesion molecule-l (ELAM-1) and the intercellular adhesion molecule-l (ICAM-1). ELAM-1 is not basally expressed on unstimulated endothelial cell surface, but it is rapidly induced by a large number of cytokines. Although its role in neutrophil adherence has been confirmed by the use of monoclonal antibodies, the specific leukocyte receptor for ELAM-1 remains undetermined [ 1, 321. ICAMis a single chain membrane adhesion glycoprotein with a low-level basal expression on vascular endothelial cells. It is also present on thymic epithelial cells, fibroblasts, and hematopoietic cells such as tissue macrophages, mitogen-stimulated T lymphocyte blasts, and germinal center dendritic cells of various lymphoid organs [33]. ICAMconsists of an integral membrane protein with an extracellular domain containing five immunoglobulin-like regions [ 1,341. It has been shown to be a ligand for the LFA-1 (CDlla/CDlB) receptor on the leukocyte cell membrane in both stimulated and unstimulated endothelial cells [l, 3, 61. Although other leukocyte adhesion molecules have been identified on endothelial cells, their role in neutrophi1 binding remains unclear. The MECA-325 antigen, present in high endothelium in mice, is induced by interferon y and has been shown to mediate lymphocyte extravasation [3]. ICAMand ELAM-2 have also been reported as possible mediators of neutrophil adherence. ICAMis believed to interact with LFA-1 [6, 71. Yet

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another adhesion molecule which binds MoAb 1.4C3 has been found on endothelial cells. It has minimal basal expression and shows maximal expression 6-10 hr after endothelial stimulation [35]. Activating

Agonists

Several agonists have been shown to act on endothelial cells to upregulate neutrophil binding. Interleukin-1 (IL-l), tumor necrosis factor-a, (TNF-cu), lymphotoxin, and bacterial endotoxin (lipopolysaccharides, LPS) induce the production of ELAM-1 and ICAMon the surface of endothelial cells, thereby increasing neutrophil adhesiveness to these cells. ELAM-1 expression is initiated 30 min after exposure to these cytokines. Its adhesive action peaks 4 hr after stimulation and declines to background levels by 24 hr despite cytokine presence. While ELAM-1 expression is still high (45%) at 24 hr, the molecule appears to have altered functional properties. In contrast, ICAMexpression increases markedly over the first 3 hr following cytokine stimulation and stays at a high level for 24 hr [l, 19, 32, 33, 36, 371. The expression of these molecules is a reversible process involving de nouo endothelial cell RNA and protein synthesis, and is thus inhibited by cycloheximide or actinomycin D [33,36]. PMN adhesion in response to IL1, TNF-a, and LPS is significantly attenuated (60-70%) by the addition of MoAb 60.3 highlighting the importance of the interaction of ICAMwith the CDwl8 (LFA-l/MAC-l/pl50, 95) neutrophil complex [36]. Increased adherence may be mediated by diverse mechanisms. Substance P has been shown to rapidly increase ELAM-1 expression on postcapillary dermal venules by mediating mast cell degranulation and release of TNF-(U [37]. Leukotriene C, and a-thrombin have been found to induce a rapid (5 min) endothelial cell-dependent increase in adherence of neutrophils. These cytokines appear to act independent of ELAM-1 and the CDll/CD18 molecule [l, 191. Monoclonal

Antibodies

and Adhesion Inhibitors

Various monoclonal antibodies to neutrophil receptors on endothelial cells have been identified. The murine MoAb H18/7 (an IgGPa) has been reported to recognize the ELAM-1 polypeptide on human endothelial cells [l, 19, 321. When administered to cytokine and endotoxin-activated endothelial cells in uitro, this MoAb reduces neutrophil adhesion by more than 50% and HL60 adhesion by more than 60% [l, 19,321. MoAb H 4/18 (IgG,, Ref. [13]) also binds to an epitope of ELAM-1, but it has no effect on neutrophil adhesion and is less effective in inhibiting HL-60 binding to activated endothelial cells [32]. Both MoAb fail to recognize cell surface antigens in unstimulated endothelial cells. Their action correlates with ELAM-1 expression, reaching maximal (65-85%) binding and neutrophil blockage 4-6 hr after cytokine treatment and decreasing to background levels

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by 24 hr [19,32]. These MoAb have not been shown to react with PMN or peripheral blood mononuclear leukocytes. Three MoAb (LB-2, 84H10, and RRl/l) have been demonstrated to block CDlla/CDlS-dependent neutrophi1 adhesion by binding to ICAMon human endothelial cells in vitro [ 7,341. This blockage is dose dependent and reaches a maximum of approximately 50% inhibition of phorbol dibutyrate-activated neutrophil binding to unstimulated endothelial cells. The number of MoAb binding sites varies from 2-35 X lo3 sites/cell in different cell cultures [7]. Recent studies have shown that diverse agents unrelated to MoAb also decrease ICAMor ELAM-1 expression. 3:Deazaadenosine (c3 Ado), a structural analog of adenosine, acts selectively on endothelial cells to reduce TNF-stimulation of ICAMto basal and possibly sub-basal levels. Human endothelial cells incubated for 4 hr in TNF and c3Ado show a time- and concentrationdependent decrease in PMN adherence. Maximal inhibition (85%) is obtained when 200 pg c3Ado is added with TNF at the beginning of the incubation period, while little inhibition occurs when the cells are incubated in TNF alone and c3Ado is added later along with the PMN. PMN binding to unstimulated endothelial cells is hardly affected by c3Ado alone [34]. Dexamethasone and hydrocortisone have been shown to inhibit IL-l induction of ICAMon human chondrosarcoma and adenocarcinoma cells, yet their actions on PMN binding to endothelial cells have not been demonstrated [38]. Transforming growth factor-p, another cytokine participating in the inflammatory response, decreases both basal and TNFa-induced PMN-endothelial cell adherence by the possible down regulation of ICAMexpression [39]. Also, interleukin 8 has been identified as an endogenous inhibitor of leukocyte adhesion and as protective agent against PMN-induced vessel wall injury [40]. CONCLUSION

It is evident that the inhibition of neutrophil adherence to microvascular endothelium can prevent neutrophi1 activation. This, in turn, leads to a reduction in PMN-mediated tissue damage. Monoclonal antibodies against neutrophil adhesion molecules have proved successful in preventing neutrophil binding and tissue injury in a variety of experimental settings [ 14,151. These studies suggest the therapeutic use of systemic MoAb to attenuate multiple organ damage after shock. Their use may also be beneficial in other acute inflammatory conditions such as adult respiratory distress syndrome or immune complex vasculitis [ 131. While these in uiuo animal studies argue for early clinical applications of MoAb therapy in humans, most work on human tissues has been limited to in vitro preparations of cultured human endothelial cells. Unfortunately, very little is known about the duration andpossi-

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ble side effects of such treatments. Perhaps the most promising avenue is direct blockage of the endothelial cell receptors (ICAMand ELAM-1) since this more localized treatment may lessen any adverse systemic effects of neutrophil inhibition. MoAb could be applied intraarterially before attempting reperfusion of an ischemit or transplanted organ. REFERENCES 1.

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