Antibodies against Adhesion Molecules Reduce Apoptosis after Transient Middle Cerebral Artery Occlusion in Rat Brain

Abstract

We tested the hypothesis that treatment of transient focal cerebral ischemia in rat with antibodies directed against adhesion molecules reduces apoptosis. Rats (n = 31) were subjected to 2 h of middle cerebral artery (MCA) occlusion induced by intraluminal insertion of a nylon monofilament into the internal carotid artery. Upon reperfusion, animals were treated with monoclonal antibodies directed against intercellular adhesion molecule (ICAM)-1) (n = 8) or integrin CD11b/CD18 (n = 10), or administered IgG1 as a control (n = 13). At 48 h after ischemia, animals were killed and the brains analyzed for ischemic cell damage, using hematoxylin and eosin (H/E); apoptosis, using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) method; and inflammatory cells, using immunohistochemistry with an anti-myeloperoxidase (MPO) antibody. Data revealed a significant reduction in the volume of infarction (p < 0.01) and a decline in the absolute (p < 0.001), and normalized (to the ischemic area, p < 0.05) numbers of apoptotic cells in both animals treated with anti-ICAM-1 and anti-CD11b antibodies compared to control animals. The numbers of immunoreactive MPO cells were also reduced in the treatment groups compared to those in the control group (p < 0.05). These data suggest that treatment with anti-adhesion molecule antibodies selectively reduce apoptosis, and that a contributing factor to the beneficial effect of antibody treatment for reducing ischemic cell damage may be a reduction in numbers of apoptotic cells.

Keywords

Apoptosis Adhesion molecules ICAM-1 CD11b Antibodies Stroke Rat

Apoptosis is present in rat brain after onset of middle cerebral artery (MCA) occlusion and may contribute to ischemic tissue damage (Linnick et al., 1993; Charriaut-Marlangue et al., 1995; Li et al., 1995a-d). Cells exhibiting apoptotic morphology undergo a process of cell death that fundamentally differs from that of necrosis. Apoptosis is orchestrated by gene and/or protein expression, in contrast to necrosis in which the initiating and promoting events are primarily associated with disruption of ionic and metabolic homeostasis (Kerr et al., 1972; Wyllie et al., 1980; Ellis et al., 1991; Bursch et al., 1992; Vaux 1993; Vaux et al., 1994; Steller (1995).

Antibodies against adhesion molecules (Clark et al., 1991a,1991b; Bowes et al., 1993; Chen et al., 1994; Chopp et al., 1994; Jiang et al., 1994; Zhang et al., 1994, 1995), as well as antibiotics (Bowes et al., 1993), and proteins (Moyle et al., 1994; Jiang et al., 1995) have been used to reduce inflammatory cell response and cell damage after experimental cerebral ischemia, inflammatory cells presumably promote ischemic cell damage by microvascular occlusion, thereby prolonging or intensifying the ischemic event, and by cytotoxic means, in which neutrophils release free radicals and proteases (Engler et al., 1983; Braide et al., 1984; Harlan 1985; Hallenbeck et al., 1986; Schmid-Schönbein and Engler, 1986; Schmid-Schönbein 1987; Granger 1988; Mullane et al., 1988; Lucchesi et al., 1989; Weiss 1989). Although the volume of cerebral infarction is reduced after administration of agents designed to reduce neutrophil adherence to endothelium and subsequent migration into the ischemic tissue, inflammatory cells may exacerbate ischemic cell damage by other means, such as by inducing apoptosis.

In the present study, we tested the effects of administration of two anti-adhesion molecule antibodies, anti-CD11b and anti-intercellular adhesion molecule (ICAM)-1, on the numbers of apoptotic cells in rats that have been subjected to transient MCA occlusion. We selected these two antibodies because they both show efficacy in reducing ischemic cell damage in the rat even when administered hours after the onset of focal cerebral ischemia (Chopp et al., 1994; Zhang RL et al., 1995; Zhang ZG et al., in press). However, these antibodies differ in that the anti-CD11b acts against a β2 integrin molecule that is primarily expressed within the leukocyte, while the anti-ICAM-1 antibody acts against a glycoprotein that is primarily expressed on the surface of the endothelial cell. Although ICAM-1 and β2 integrins induce apoptosis in T cells (Damle et al., 1993), to our knowledge, there have been no studies on the role of postischemic inflammatory cells and adhesion molecules as mediators of apoptosis.

METHODS

Male Wistar rats (weighing 270–300 g, n = 31) were used in the experiments. Transient MCA occlusion was induced by advancing a 4–0 surgical nylon monofilament into the internal carotid artery (ICA) to block the origin of the MCA (Zea Longa et al., 1989; Chen et al., 1992). Briefly, animals were anesthetized with 3.5% halothane, and anesthesia was maintained with 1.0% halothane in 70% N₂O and 30% O₂ (vol/vol) using a face mask. Rectal temperature was maintained at 37°C throughout the surgical procedure using a feedback-regulated water heating system. The right femoral artery and vein were cannulated for measuring blood gases (pH, Po₂, Pco₂) before ischemia and for drug administration, respectively. A length of 18.5–19.0 mm 4–0 surgical nylon filament with its tip rounded by heating near a flame, was advanced from the external carotid artery into the lumen of the ICA until it blocked the origin of the MCA. At 2 h after MCA occlusion, animals were reanesthetized with halothane and reperfusion was performed by withdrawal of the filament until the tip cleared the ICA lumen.

Animals were divided into three groups. Anti-ICAM-1 group (n = 8) animals were subjected to MCA occlusion and an anti-rat ICAM-1 antibody was infused (i.v.) over a 3-min interval at a dose of 1 mg/kg upon reperfusion and 0.5 mg/kg at 22 h of reperfusion. Anti-CD11b group (n = 10) animals were subjected to MCA occlusion and anti-CD11b antibody was infused (i.v.) over a 3-min interval at a dose of 2 mg/kg upon reperfusion and 1.0 mg/kg at 22 h of reperfusion. Control group (n = 13) animals were subjected to MCA occlusion and an isotype-matched control antibody (mouse IgG1, Sigma Chemicals, St. Louis, MO, U.S.A.) was administered upon reperfusion and at 22 h of reperfusion, using the same volume dose as the experimental groups. The treatment antibodies have endotoxin levels of < 1.0 eu/mg.

The numbers of apoptotic cells within ischemic rat brain subjected to 2 h of MCA occlusion are maximum at 48 h after onset of ischemia in rats (Li et al., 1995a). Although it is preferable to kill animals at times >48 h when infarct volumes are to be measured, the primary hypothesis to be tested was that antibodies against adhesion molecules reduce numbers of apoptotic cells in ischemic tissue; therefore, 48 hour was selected as the most efficacious time for counting of apoptotic cells.

Antibody to rat ICAM-1, designated 1A29, was developed according to details provided in a recent publication (Tamatani and Miyasaka 1993) and was obtained from Drs. Anthony Manning and Donald Anderson at the Upjohn Company (Kalamazoo, MI, U.S.A.). The anti-CD11b antibody (clone 1B6c, F(ab′)₂) was obtained from Repligen Corporation (Cambridge, MA, U.S.A.), and is a mouse anti-rat antibody (Mulligan et al., 1992, 1993; Zhang ZG et al., in press).

Animals were anesthetized (i.m.) with ketamine (44 mg/kg/body wt) and xylazine (13 mg/kg/body wt) at 46 h of reperfusion. Rats were transcardially perfused with heparinized saline and 10% buffered formalin, and brains were removed. Each brain was cut into seven 2 mm-thick coronal blocks (A-G), using a rat brain matrix. Brain tissue was processed and embedded, and 5 μm-thick paraffin sections were cut from each block and stained with hematoxylin and eosin (H/E) for histopathological evaluation. In addition, adjacent 5 μm-thick paraffin sections were obtained from block D and a molecular biological-histochemical system (ApopTag kit, Oncor, Gaithersburg, MD, U.S.A.) for specific staining of DNA fragmentation and apoptotic bodies, the terminal deoxynucleotidyl (TdT)-mediated dUTP biotin nick end labeling (TUNEL) stain (Gavrieli et al., 1992), and immunohistochemistry of myeloperoxidase (MPO) for neutrophil detection (Daugherty et al., 1994) were employed.

The area of infarction was measured using a Global Lab Image analysis program (Data Translation, Malboro, MA, U.S.A.). Each H/E section was evaluated at ×2.5 magnification. The area of infarction and the area of the ipsilateral hemisphere (mm²) were calculated by tracing the areas on the computer screen. To reduce errors associated with the processing of tissue for histological analysis, the area of infarction in each section was presented as the percentage of the infarct to the ipsilateral hemispheric area; the infarct volume was also presented as the percentage of infarct to the ipsilateral hemisphere.

Cells exhibiting DNA fragmentation were counted in both the ipsilateral and contralateral hemispheres using a light microscope (×200–300 magnification). Cells containing apoptotic bodies are referred to as apoptotic cells (Gavrieli et al., 1992; Wijsman et al., 1993; Wood et al., 1993; Li et al., 1995a). The TUNEL method is based on the specific binding of TdT to the 3′-OH ends of DNA and the ensuing synthesis of a polydeoxynucleotide polymer. Briefly, after deparaffinizing the brain sections, digesting the protein in specimens with proteinase K, then quenching endogenous peroxidase activity with 2% H₂O₂ in phosphate-buffered saline (PBS), slides were placed in equilibration buffer and then in TdT enzyme, followed by stop/wash buffer. After two drops of anti-digoxigenin-peroxidase was applied to the slides, peroxidase was detected with diaminobenzidine. Negative controls were performed using distilled water to replace TdT Enzyme. The labeling targets of the TUNEL staining were the new 3′-OH DNA ends generated by DNA fragmentation; these were typically localized in morphologically identifiable nuclei and apoptotic bodies. The direct immuno-peroxidase detection of digoxigenin-labeled DNA in thin sections of fixed tissue allowed analysis of scattered cells exhibiting DNA fragmentation at anatomical locations after MCA occlusion. In contrast, normal nuclei, which had relatively insignificant numbers of DNA 3′-OH ends, did not stain with the TUNEL method. Cells exhibiting necrotic morphologies contained, in some instances, stain-able concentrations of DNA ends; however, staining in necrotic cells appeared more diffuse than in apoptotic cells. Coronal sections stained with the TUNEL method were also counterstained with hematoxylin.

Numbers of neutrophils within brain were counted using immunohistochemical staining for MPO (Bradley et al., 1982; Daugherty et al., 1994). MPO is a marker for inflammatory cells, primarily neutrophils (Barone et al., 1991). Sections (5 UJTI thick) adjacent to those used for counting apoptotic cells were obtained and stained with a polyclonal rabbit anti-human MPO antibody (Dako no. A398, Copenhagen, Denmark). All immunoreactive cells were counted within the ipsilateral hemisphere using a light microscope (magnification ×132).

Welch's two-sample t-tests were used to compare the anti-ICAM-1 and anti-CD11b groups to the vehicle group with respect to lesion area, ratio of lesion area/ipsilateral hemisphere area, number of apoptotic cells, and ratio of the number of apoptotic cells to the lesion area. A Bonferroni-adjusted α-level of 0.025 (0.05/2) was used to interpret results of each test. P-values of ≦0.025 were considered to be statistically significant, while p-values of 0.025–0.05 were considered to be “marginally” significant. All measurements were performed blindly (by J.P., N.J., R.L.Z., Y.L.). All data are presented as mean value + SD.

RESULTS

Blood gases were within normal ranges for all animals and did not differ between groups (Table 1). Animals treated with anti-ICAM-1 and anti-CD11b antibodies exhibited percent cerebral infarct volumes of 18.0 ± 11.2 (n = 8) and 24.3 ± 11.0 (n = 10), respectively, that were significantly smaller (p = 0.01) than the percent cerebral infarct volumes obtained in control animals (36.5 ± 7.7; n = 13). These data are consistent with those of our previous reports (Chen et al., 1994; Jiang et al., 1994; Zhang et al., 1994).

TABLE 1.

Blood gases (mean ± SD) in the three groups

Groups^a	pH	pCO₂ (mm Hg)	pO₂ (mm Hg)
Control (n = 13)	7.40 ± 0.04	38.1 ± 6.48	120.6 ± 11.2
ICAM-1 (n = 8)	7.39 ± 0.05	37.5 ± 4.42	115.5 ± 23.5
CD11b (n = 10)	7.41 ± 0.04	39.6 ± 9.70	124.4 ± 19.9

Groups are defined in text.

Figure 1 shows a representative coronal section of striatal tissue from a control animal in which apoptotic and inflammatory cells are readily apparent within the ischemic region. As we have previously reported in control ischemic animals (Li et al., 1995a –Li et al., 1995c), the apoptotic cells are primarily concentrated along the inner boundary of the zone of infarction.

FIG. 1.

Coronal sections (5 μm thick) stained with hematoxylin and eosin from striatal tissue of an animal subjected to 2h of MCA occlusion and sacrificed 48 h after onset of reperfusion. ↑, neutrophil; ⇑, apoptotic cell; ▾, necrotic neuron original magnification ×660.

The difference in the mean number of apoptotic cells was found to be significantly different between vehicle (186.3 ± 43.4; n = 13) and anti-ICAM-1 (54.8 ± 57.0; n = 8) (p < 0.001), and vehicle and anti-CD11b (79.3 ± 57.1; n = 10) (p < 0.001). For the normalized numbers of apoptotic cells, the vehicle group (7.6 ± 2.6; n = 13) was found to be significantly different from the anti-ICAM-1 (4.2 ± 3.2; n = 8) (p = 0.023), and the vehicle group was marginally significantly different from the anti-CD11b (4.9 ± 3.1; n = 10) treatment group (p = 0.040).

The number and ischemic area normalized numbers of MPO immunoreactive cells were significantly (p < 0.01) reduced within the treated groups compared to the control group: control group (596 ± 331; 23.6 ± 11.6 mm⁻²; n = 5); anti-ICAM-1 group (166 ± 144; 13.8 ± 8.3 mm⁻²; n = 6); and anti-CD11b group (170 ± 142; 16.1 ± 10.5 mm⁻⁵; n = 5). No difference was detected in the absolute and normalized numbers of MPO immunoreactive cells between the anti-ICAM-1 and anti-CD11b groups. Numbers of immunoreactive MPO and apoptotic cells within adjacent sections of tissue were highly correlated (r = 0.78; p < 0.001). Figure 2 illustrates the relative distribution of apoptotic and immunoreactive MPO cells within a coronal section. Immunoreactive MPO cells exhibited a similar spatial pattern of distribution as the apoptotic cells.

FIG. 2.

Schematic illustration of the spatial distribution of apoptotic: (⋄) and MPO (•) immunoreactive cells within a coronal section (D) of cerebral tissue. The gray shading corresponds to neuronal necrosis.

DISCUSSION

Our data indicate that administration of antibodies designed to reduce leukocyte adhesion and subsequent migration into ischemic tissue also reduces the numbers of apoptotic cells. There is a significant attenuation of apoptotic cells (absolute and normalized numbers) after treatment designed to reduce postischemic inflammatory response. Thus, the therapeutic benefit of administration of antibodies against adhesion molecules not only is associated with improvement of cerebral blood flow and reduction of cytotoxicity but also includes a reduction in the number of cells undergoing apoptosis.

There are several possible explanations for the reduction of apoptotic cells after treatment with anti-adhesion molecule antibodies. Reduction of apoptosis can be attributed to a reduction in numbers of inflammatory cells and/or to a direct effect of the antibodies on parenchymal cells. Inflammatory cells can induce apoptosis (Squier et al., 1995). Cytotoxic T lymphocytes undergo apoptosis and induce apoptosis in target cells (Duke et al., 1983; Ucker, 1987; Sellins and Cohen 1991; Nagata and Golstein, 1995). Although neutrophils undergo apoptosis (Squier et al., 1995), to date, there are no reports that neutrophils directly induce apoptosis in target cells. However, it is possible that the reduction in numbers of neutrophils within ischemic tissue indirectly causes reduction of apoptosis. This hypothesis is consistent with the robust correlation of immunoreactive MPO and apoptotic cells as well as the similar spatial distribution of immunoreactive MPO and apoptotic cells. Neutrophils are a potent source of oxygen-free radicals, and free radicals induce apoptosis (Hockenbery et al., 1991, 1993). Adherent neutrophils can release oxidizing radicals into the neural cell cytoplasm. Proteases, likewise, induce apoptosis (Sarin et al., 1993; Miura et al., 1994; Thompson, 1995), and leukocytes are a profuse source of proteases (Henson and Johnston, 1987; Weiss, 1989; Hansen, 1995). Expression of matrix-degrading proteases in normal tissues causes apoptosis (Talhouk et al., 1992). Interleukin 1β-converting enzyme (ICE) is a protease that facilitates apoptosis in mammalian cell lines (Los et al., 1995), and inflammatory cells express proteases, such as granzyme β, with similar apoptotic activity (Sleath et al., 1990; Howard et al., 1991; Odake et al., 1991; Shi et al., 1992; Caputo et al., 1994; Heusel et al., 1994).

Reduction of apoptotic cells compared to vehicle-treated animals was more pronounced with anti-ICAM-1 antibody treatment (p = 0.023) than with anti-CD11b antibody treatment (p = 0.040). However, the number of neutrophils within the ischemic tissue was nearly identical for the anti-ICAM-1 and anti-CD11b-treated groups; if apoptosis is caused by a direct attack of neutrophils on the parenchymal cells, then we expect the numbers of apoptotic cells to closely reflect the numbers of neutrophils within the tissue. It is, therefore, unclear why blocking the ICAM-1 sites shows a trend (although not statistically significant) towards being more effective in reducing apoptosis than blocking the CD11b sites. A possible explanation for the more potent anti-ICAM-1 reduction of apoptosis is that cell adhesion molecules (CAMs) directly affect neurons. During development of the nervous system, CAMs promote neuronal migration and axonal growth; yet, at other times, CAMs inhibit these events by maintaining stable adhesion between cells (Doherty and Walsh, 1994). Cytokines released in the course of inflammation induce expression of ICAM-1 on neurons, allowing them to be targeted by neutrophils (Birdsall, 1991). Adherence of neutrophils to cytokine-stimulated neural cells is mediated primarily by ICAM-1 and lymphocyte function antigen-I (LFA-1) interactions, and 70–90% of the binding can be blocked by monoclonal antibodies to either ligand (Birdsall, 1991). A direct role for adhesion molecule mediated-apoptosis was recently demonstrated in which T-cell receptor-directed engagement of integrins by their ligands ICAM-1 or VCAM-1 induced apoptosis of T cells (Damle et al., 1993).

Although the mechanism by which the anti-ICAM-1 antibody inhibits apoptosis is not known, it may be related to a coupling of ICAM-1, IL-1β, and ICE. The mammalian gene, ICE, encodes a cysteine protease involved in the processing of IL-1β. ICE is required for the conversion of pro IL-1β into active IL-1β. ICE induces apoptosis and activates IL-1β, which, in turn, induces ICAM-1 (Springer, 1994). Conversely, whether blocking ICAM-1 inhibits IL-1β and ICE, and, subsequently, reduces apoptosis is not known. An additional pathway relating adhesion molecules and apoptosis is related to the observation that tumor suppressor genes encode cell adhesion molecules (Hedrick et al., 1993). Tumor suppressor genes, e.g. p53, also induce apoptosis (Clarke et al., 1993). Thus, apoptosis may be facilitated by adhesion molecules, and, therefore, blocking adhesion molecules may reduce apoptosis, as demonstrated in the present study.

We have employed an immunohistochemical method using an anti-MPO antibody to measure inflammatory cells within the ischemic brain. This method has recently been applied to studies of human atherosclerotic lesions (Daugherty et al., 1994), and we adapted this method to the study of brain. MPO is a gold standard measurement for the presence of neutrophils (Barone et al., 1991). Although monocytes also possess MPO activity, MPO in monocytes accounts for <1% of the MPO recovered from inflammatory skin lesions (Bradley et al., 1982). We cannot exclude the possibility that other cells, such as monocytes and macrophages, may be included in the total MPO positive cell count.

The cellular target for the anti-adhesion molecule antibodies is not restricted to neutrophils, but may also include other exogenous inflammatory cells, such as monocytes and T lymphocytes. The antibodies employed in the present study are effective on different leukocytes. The CD11b receptor is most prominently expressed on neutrophils and monocytes (Kishimoto et al., 1989). In contrast, the ICAM-1 is a ligand for multiple β2 integrins, including those such as LFA-1, that are expressed on lymphocytes (Rothlein et al., 1986; Simmons et al., 1988; Staunton et al., 1988). ICAM-1 is also expressed in primary cultures of rat astrocytes (Shrikant et al., 1994). Although the focus of our study was apoptosis and MPO immunoreactive cells and although we did not measure the temporal profile of lymphocytes in ischemic tissue in the present study, T lymphocytes were present. Figure 3 shows a T lymphocyte within the ischemic tissue at 48 h after onset of reperfusion. It is, thus, possible, and further study is required to demonstrate, that the ability of the anti-adhesion molecule antibodies to reduce apoptosis may be related to the effect of antibodies, particularly ICAM-1, which is a ligand for LFA-1, on T lymphocytes or other inflammatory cells.

FIG. 3.

Coronal sections (5 μm thick), stained with hematoxylin and eosin, obtained from ischemic brain from a control animal. T-lymphocyte (↑) is present within the ischemic tissue. Original magnification ×660.

In summary, we have demonstrated that antibodies directed against CD11b and ICAM-1 adhesion molecules suppress apoptosis in ischemic brain. This observation raises the intriguing possibility that inflammatory cells and/or adhesion molecules and receptors promote apoptosis, and that antibodies against adhesion molecules suppress apoptosis in ischemic brain.

Footnotes

Acknowledgment:

The authors are grateful to Dr. Zhang G. Zheng for helpful discussions and technical assistance and Denice Janus for secretarial support. This work was supported in part from NINDS grants PO1 NS23393, RO1 NS33627, and RO1 NS34184.

Abbreviations used

References

Barone

Hillegass

Price

White

Lee

Feuerstein

Sarau

Clark

Griswold

(1991) Polymorphonuclear leukocyte infiltration into cerebral focal ischemic tissue: Myeloperoxidase activity assay and histologic verification. J Neurol Res 29:336–345

Birdsall

(1991) Induction of ICAM-1 on human neural cells and mechanisms of neutrophil-mediated injury. Am J Pathol 139:1341–1350

Bowes

Zivin

Rothlein

(1993) Monoclonal antibody to the ICAM-1 adhesion site reduces neurological damage in a rabbit cerebral embolism stroke model. Exp Model 119:215–219

Bradley

Priebat

Christensen

Rothstein

(1982) Measurement of cutaneous inflammation: Estimation of neutrophil content with an enzyme marker. J Invest Dermatol 78:206–209

Braide

Amundson

Chien

Bagge

(1984) Quantitative studies on the influence on leukocytes on the vascular resistance in a skeletal muscle preparation. Microvasc Res 27: 331–352

Bursch

Oberhammer

Schulte-Hermann

(1992) Cell death by apoptosis and its protective role against disease. Trends Pharmacol Sci 13:245–251

Caputo

James

MNG

Powers

Hudig

Bleackley

(1994) Conversion of the substrate specificity of mouse proteinase granzyme. Nat Struct Biol 1:364–367

Charriaut-Marlangue

Margaill

Plotkine

Ben-Ari

(1995) Early endonuclease activation following reversible focal cerebral ischemia in the rat brain. J Cereb Blood Flow Metab 15:385–388

Chen

Chopp

Zhang

Garcia

(1992) The effect of hypothermia on the transient cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 12:621–628

10.

Chen

Chopp

Zhang

Bodzin

Chen

Rusche

Todd

III (1994) Anti-CD11b monoclonal antibody reduces ischemic cell damage after transient focal cerebral ischemia in rat. Ann Neurol 35:447–452

11.

Chopp

Zhang

Chen

Jiang

Rusche

(1994) Post ischemic administration of an anti-Mac-1 antibody reduces ischemic cell damage after transient middle cerebral artery occlusion in the rat. Stroke 25:869–876

12.

Clark

Madden

Rothlein

Zivin

(1991a) Reduction of central nervous system ischemic injury by monoclonal antibody to intercellular adhesion molecule. J Neurosurg 75:623–627

13.

Clark

Madden

Rothlein

Zivin

(1991b) Reduction of central nervous system ischemic injury in rabbits using leukocyte adhesion antibody treatment. Stroke 22:877–883

14.

Clarke

Purdie

Harrison

Morris

Bird

Hooper

Wyllie

(1993) Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature 362:849–852

15.

Damle

Klussman

Leytze

Arruffo

Linsley

Ledbetter

(1993) Costimulation with integrin ligands intercellular adhesion molecule-1 or vascular cell adhesion molecule-1 augments activation-induced death of antigen-specific CD4⁺ T lymphocytes. J Immunol 11:2368–2379

16.

Daugherty

Dunn

Rateri

Heinecke

(1994) Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions. J Clin Invest 94:437–444

17.

Doherty

Walsh

(1994) Signal transduction events underlying neurite outgrowth stimulated by cell adhesion molecules. Curr Opinion Neurobiol 4:49–55

18.

Duke

Chervenak

Cohen

(1983) Endogenous endonuclease-induced DNA fragmentation: An early event in cell-mediated cytolysis. Proc Natl Acad Sci USA 80:6361–6365

19.

Ellis

Yuan

Horvitz

(1991) Mechanisms and functions of cell death. Ann Rev Cell Biol 7:663–698

20.

Engler

Schmid-Schönbein

Pavelec

(1983) Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog. Am J Pathol 111:98–111

21.

Gavrieli

Sherman

Ben-Sasson

(1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493–501

22.

Granger

(1988) Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury. Am J Physiol 255:H1269–H1275

23.

Hallenbeck

Dutka

MAJ

Tanishima

Kochanek

Kumaroo

Thompson

Obrenovitch

Contreras

(1986) Polymorphonuclear leukocyte accumulation in brain regions with low blood flow during the early postischemic period. Stroke 17:246–253

24.

Hansen

(1995) Role of neutrophil in myocardial ischemia and reperfusion. Circulation 91:1872–1885

25.

Harlan

(1985) Leukocyte-endothelial interaction. Blood 65: 513–525

26.

Hedrick

Cho

Vogelstein

(1993) Cell adhesion molecules as tumor suppressors. Trends Cell Biol 3:36–39

27.

Henson

Johnston

(1987) Tissue injury in inflammation. J Clin Invest 79:669–674

28.

Heusel

Wesselschmidt

Shresta

Russell

Ley

(1994) Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells. Cell 76:977–987

29.

Hockenbery

Oltvai

Yin

X-M

Milliman

Korsmeyer

(1993) Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75:241–251

30.

Hockenbery

Zutter

Hickey

Nahm

Korsmeyer

(1991) Bcl-2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci USA 88:6961–6965

31.

Howard

Kostura

Thornberry

Ding

Limjuco

Weidner

Salley

Hogquist

Chaplin

Mumford

Schmidt

Tocci

(1991) IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1β precursor at two distinct sites and does not cleave 31-kDa IL-1α. J Immunol 147:2964–2969

32.

Jiang

Zhang

Chen

Chopp

(1994) Anti-CD11b monoclonal antibody reduces ischemic cell damage after transient (2h) but not after permanent MCA occlusion in the rat. Neurosci Res Comm 15:85–94

33.

Jiang

Moyle

Soule

Rote

Chopp

(1995) Postischemic administration of the CD11b/CD18 antagonist neutrophil inhibitory factor (NIF) is neuroprotective after transient middle cerebral artery occlusion in rats. Ann Neurol (in press)

34.

Kerr

JFR

Wyllie

Currie

(1972) Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239–257

35.

Kishimoto

Larson

Corbi

Dustin

Staunton

Springer

(1989) The leukocyte integrins. Adv Immunol 46:149–182

36.

Chopp

Jiang

Yao

Zaloga

(1995a) Temporal profile of in situ DNA fragmentation after transient middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 15:389–397

37.

Chopp

Jiang

Zaloga

(1995b) In situ detection of DNA fragmentation after focal cerebral ischemia in mice. Mol Brain Res 28:164–168

38.

Chopp

Jiang

Zhang

Zaloga

(1995c) Induction of DNA fragmentation after 10–120 minutes of focal cerebral ischemia in rat. Stroke 26:1252–1258

39.

Sharov

Jiang

Yao

Zaloga

Sabbah

Chopp

(1995d) Ultrastructural and light microscopic evidence of apoptosis after middle cerebral artery occlusion in rat. Am J Pathol 146:1045–1051

40.

Linnick

Zobrist

Hatfield

(1993) Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats. Stroke 24:2002–2009

41.

Los

Van de Craen

Penning

Schenk

Westendorp

Baeuerle

Droge

Krammer

Fiers

Schulze-Osthoff

(1995) Requirement of an ICE/CED-3 protease for Fas/APO-1 mediated apoptosis. Nature 375:81–83

42.

Lucchesi

Werns

Fantone

(1989) The role of the neutrophil and free radicals in ischemic myocardial injury. J Mol Cell Cardiol 21:1241–1261

43.

Miura

Zhu

Rotello

Hartwieg

Yuan

(1994) Induction of apoptosis in fibroblasts by IL-IB-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell 75:653–660

44.

Moyle

Foster

McGrath

Brown

Laroche

DeMeutter

Stanssens

Bogowitz

Fried

Ely

Soule

Vlasuk

(1994) A hookworm glycoprotein that inhibits neutrophil function is a ligand of the integrin CD11b/CD18. J Biol Chem 269:10008–10015

45.

Mullane

Westlin

Kraemer

(1988) Activated neutrophils release mediators that may contribute to myocardial dysfunction associated with ischemia and reperfusion. Ann NY Acad Sci 524:103–121

46.

Mulligan

Varani

Warren

Till

Smith

Anderson

Todd

III Ward

(1992) Role of beta-2 integrins of rat neutrophils in complement- and oxygen radical-mediated acute inflammatory injury. J Immunol 149:1847–1857

47.

Mulligan

Wilson

Todd

III Rusche

Smith

Anderson

Varani

Issekutz

Miyasaka

Tamatani

Ward

(1993) Role of B_b B₂ integrins and ICAM-1 in lung injury following deposition of IgG and IgA immune complexes. J Immunol 150:2407–2417

48.

Nagata

Golstein

(1995) The Fas death factor. Science 267: 1449–1455

49.

Odake

Kam

Narasimhan

Poe

Blake

Krahenbuhl

Tschopp

Powers

(1991) Human and murine cytotoxic T lymphocyte serine proteases: Subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins. Biochemistry 30: 2217–2227

50.

Rothlein

Dustin

Marlin

Springer

(1986) A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1. J Immunol 137:1270–1274

51.

Sarin

Adams

Henkart

(1993) Protease inhibitors selectively block T cell receptor triggered programmed cell death in a murine T cell hybridoma and activated peripheral T cells. J Exp Med 178:1693–1700

52.

Schmid-Schönbein

Engler

(1986) Granulocytes as active participants in acute myocardial ischemia and infarction. Am J Cardiovasc Pathol 1:15–30

53.

Schmid-Schönbein

(1987) Capillary plugging by granulocytes and the no-reflow phenomenon in the microcirculation. Proc Fed Am Soc Exp Biol 46:2397–2401

54.

Sellins

Cohen

(1991) Cytotoxic T lymphocytes induce different types of DNA damage in target cells of different origins. J Immunol 147:795–803

55.

Shi

Kam

Powers

Aebersold

Greenberg

(1992) Purification of three cytotoxic lymphocyte granule serine proteases that induce apoptosis through distinct substrate and target cell interactions. J Exp Med 176:1521–1529

56.

Shrikant

Chung

Ballestas

Benveniste

(1994) Regulation of intercellular adhesion molecule-1 gene expression by tumor necrosis factor-α, interleukin-1β, and interferon-γ in astrocytes. J Neuroimmunol 51:209–220

57.

Simmons

Makgoba

Seed

(1988) ICAM, an adhesion ligand of LFA-1 is homologous to the neural cell adhesion molecule NCAM. Nature 331:624–627

58.

Sleath

Hendrickson

Kronheim

March

Black

(1990) Substrate specificity of the protease that processes human interleukin-1 beta. J Biol Chem 265:14526–14528

59.

Springer

(1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 76: 301–314

60.

Squier

MKT

Sehnert

Cohen

(1995) Apoptosis in leukocytes. J Leuk Biol 57:2–10

61.

Staunton

Marlin

Stratowa

Dustin

Springer

(1988) Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families. Cell 52:925–933

62.

Steller

(1995) Mechanisms and genes of cellular suicide. Science 267:1445–1449

63.

Talhouk

Bisseil

Werb

(1992) Coordinated expression of extracellular matrix degrading proteinases and their inhibitors regulates mammary epithelial function during involution. J Cell Biol 118:1271–1282

64.

Tamatani

Miyasaka

(1993) Identification of monoclonal antibody reactive with the rat homologue of ICAM-1, and evidence for differential involvement of ICAM-1 in the adherence of resting versus activated lymphocytes to high endothelial cells. Int Immunol 2:166–172

65.

Thompson

(1995) Apoptosis in the pathogenesis and treatment of disease. Science 267:1456–1461

66.

Ucker

(1987) Cytotoxic T lymphocytes and glucocortoids activate an endogenous suicide process in target cells. Nature 327:62–64

67.

Vaux

(1993) Toward an understanding of the molecular mechanisms of physiological cell death. Proc Natl Acad Sci USA 90:786–789

68.

Vaux

Haecker

Strasser

(1994) An evolutionary perspective on apoptosis. Cell 76:777–779

69.

Weiss

(1989) Tissue destruction by neutrophils. N Engl J Med 320:365–375

70.

Wijsman

Jonker

Keijzer

Ven de Velde

CJH

Cornelisse

Van Dierendonck

(1993) A new method to detect apoptosis in paraffin sections: In situ end-labeling of fragmented DNA. J Histochem Cytochem 41:7–12

71.

Wood

Dipasquale

Youle

(1993) In situ labeling of granule cells for apoptosis-associated DNA fragmentation reveals different mechanisms of cell loss in developing cerebellum. Neuron 11:621–632

72.

Wyllie

Kerr

JFR

Currie

(1980) Cell death: The significance of apoptosis. Int Rev Cytol 68:251–306

73.

Zea Longa

Weinstein

Carlson

Cummins

(1989) Reversible middle cerebral artery occlusion with craniectomy in rats. Stroke 20:84–89

74.

Zhang

Chopp

Zaloga

Jiang

Jones

Miyasaka

Ward

(1994) Anti-ICAM-1 antibody reduces ischemic cell damage after transient middle cerebral artery occlusion in the rat. Neurology 44:1747–1751

75.

Zhang

Chopp

Jiang

Tang

Prostak

Manning

(1995) Anti-ICAM-1 antibody reduces ischemic cell damage after transient but not permanent MCA occlusion in the Wistar rat. Stroke 26:1438–1443

76.

Zhang

Chopp

Tang

Jiang

Zhang

(1995) Postischemic treatment (24h) with anti-CD11b and anti-CD18 monoclonal antibodies are neuroprotective after transient (2h) focal cerebral ischemia in the rat. Brain Res (in press)