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

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.

[1]  C. Thompson,et al.  Apoptosis in the pathogenesis and treatment of disease , 1995, Science.

[2]  J. Weidner,et al.  IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha. , 1991, Journal of immunology.

[3]  M. Chopp,et al.  Induction of DNA fragmentation after 10 to 120 minutes of focal cerebral ischemia in rats. , 1995, Stroke.

[4]  M. Linnik,et al.  Evidence Supporting a Role for Programmed Cell Death in Focal Cerebral Ischemia in Rats , 1993, Stroke.

[5]  S. Weiss.  Tissue destruction by neutrophils. , 1989, The New England journal of medicine.

[6]  V. Fried,et al.  A hookworm glycoprotein that inhibits neutrophil function is a ligand of the integrin CD11b/CD18. , 1994, The Journal of biological chemistry.

[7]  T. Springer.  Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm , 1994, Cell.

[8]  R. Aebersold,et al.  Purification of three cytotoxic lymphocyte granule serine proteases that induce apoptosis through distinct substrate and target cell interactions , 1992, The Journal of experimental medicine.

[9]  R. Rothlein,et al.  Monoclonal Antibody to the ICAM-1 Adhesion Site Reduces Neurological Damage in a Rabbit Cerebral Embolism Stroke Model , 1993, Experimental Neurology.

[10]  G. Schmid-Schönbein,et al.  Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog. , 1983, The American journal of pathology.

[11]  S. Sharar,et al.  Leukocyte-endothelial interactions and organ injury , 1994 .

[12]  M. Chopp,et al.  Ultrastructural and light microscopic evidence of apoptosis after middle cerebral artery occlusion in the rat. , 1995, The American journal of pathology.

[13]  P. Linsley,et al.  Costimulation with integrin ligands intercellular adhesion molecule-1 or vascular cell adhesion molecule-1 augments activation-induced death of antigen-specific CD4+ T lymphocytes. , 1993, Journal of immunology.

[14]  Junying Yuan,et al.  Induction of apoptosis in fibroblasts by IL-1β-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3 , 1993, Cell.

[15]  G. Schmid-Schönbein.  Capillary plugging by granulocytes and the no-reflow phenomenon in the microcirculation. , 1987, Federation proceedings.

[16]  B. Seed,et al.  ICAM, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM , 1988, Nature.

[17]  Y. Ben-Ari,et al.  Early Endonuclease Activation following Reversible Focal Ischemia in the Rat Brain , 1995, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[18]  Timothy J. Ley,et al.  Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells , 1994, Cell.

[19]  Michael Loran Dustin,et al.  A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1. , 1986, Journal of immunology.

[20]  J. Cohen,et al.  Apoptosis in leukocytes , 1995, Journal of leukocyte biology.

[21]  C. Smith,et al.  Roles of beta 2 integrins of rat neutrophils in complement- and oxygen radical-mediated acute inflammatory injury. , 1992, Journal of immunology.

[22]  M Chopp,et al.  Anti-intercellular adhesion molecule-1 antibody reduces ischemic cell damage after transient but not permanent middle cerebral artery occlusion in the Wistar rat. , 1995, Stroke.

[23]  A. Wyllie,et al.  Cell death: the significance of apoptosis. , 1980, International review of cytology.

[24]  M. Chopp,et al.  In situ detection of DNA fragmentation after focal cerebral ischemia in mice. , 1995, Brain Research. Molecular Brain Research.

[25]  S. Nagata,et al.  The Fas death factor , 1995, Science.

[26]  H. Sarau,et al.  Polymorphonuclear leukocyte infiltration into cerebral focal ischemic tissue: Myeloperoxidase activity assay and histologic verification , 1991, Journal of neuroscience research.

[27]  Richard S. Larson,et al.  The leukocyte integrins. , 1989, Advances in immunology.

[28]  D. Adams,et al.  Protease inhibitors selectively block T cell receptor-triggered programmed cell death in a murine T cell hybridoma and activated peripheral T cells , 1993, The Journal of experimental medicine.

[29]  M. Chopp,et al.  Postischemic Administration of an Anti‐Mac‐1 Antibody Reduces Ischemic Cell Damage After Transient Middle Cerebral Artery Occlusion in Rats , 1994, Stroke.

[30]  G. Schmid-Schönbein,et al.  Granulocytes as active participants in acute myocardial ischemia and infarction. , 1987, The American journal of cardiovascular pathology.

[31]  J. Cohen,et al.  Cytotoxic T lymphocytes induce different types of DNA damage in target cells of different origins. , 1991, Journal of immunology.

[32]  R. Black,et al.  Substrate specificity of the protease that processes human interleukin-1 beta. , 1990, The Journal of biological chemistry.

[33]  P. Weinstein,et al.  Reversible middle cerebral artery occlusion without craniectomy in rats. , 1989, Stroke.

[34]  W. Fiers,et al.  Requirement of an ICE/CED-3 protease for Fas/APO-1-mediated apoptosis , 1995, Nature.

[35]  A. Daugherty,et al.  Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions. , 1994, The Journal of clinical investigation.

[36]  Z. Oltvai,et al.  Bcl-2 functions in an antioxidant pathway to prevent apoptosis , 1993, Cell.

[37]  P. Hansen,et al.  Role of neutrophils in myocardial ischemia and reperfusion. , 1995, Circulation.

[38]  A. Bretscher,et al.  Identification of ezrin as an 81-kDa tyrosine-phosphorylated protein in T cells. , 1992, Journal of Immunology.

[39]  Michael Chopp,et al.  Postischemic treatment (2–4 h) with anti-CD11b and anti-CD18 monoclonal antibodies are neuroprotective after transient (2 h) focal cerebral ischemia in the rat , 1995, Brain Research.

[40]  David L. Vaux,et al.  An evolutionary perspective on apoptosis , 1994, Cell.

[41]  F. Walsh,et al.  Signal transduction events underlying neurite outgrowth stimulated by cell adhesion molecules , 1994, Current Opinion in Neurobiology.

[42]  H. Horvitz,et al.  Mechanisms and functions of cell death. , 1991, Annual review of cell biology.

[43]  M Chopp,et al.  The Effect of Hypothermia on Transient Middle Cerebral Artery Occlusion in the Rat , 1992, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[44]  B. Vogelstein,et al.  Cell adhesion molecules as tumour suppressors. , 1993, Trends in cell biology.

[45]  D. Ucker.  Cytotoxic T lymphocytes and glucocorticoids activate an endogenous suicide process in target cells , 1987, Nature.

[46]  D. Granger,et al.  Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury. , 1988, The American journal of physiology.

[47]  M. Ballestas,et al.  Regulation of intercellular adhesion molecule-1 gene expression by tumor necrosis factor-α, interleukin-1β, and interferon-γ in astrocytes , 1994, Journal of Neuroimmunology.

[48]  J. Harlan.  Leukocyte-endothelial interactions. , 1985, Blood.

[49]  R. Youle,et al.  In situ labeling of granule cells for apoptosis-associated DNA fragmentation reveals different mechanisms of cell loss in developing cerebellum , 1993, Neuron.

[50]  A. Wyllie,et al.  Apoptosis: A Basic Biological Phenomenon with Wide-ranging Implications in Tissue Kinetics , 1972, British Journal of Cancer.

[51]  J. H. Dierendonck,et al.  A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA. , 1993, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[52]  P. Henson,et al.  Tissue injury in inflammation. Oxidants, proteinases, and cationic proteins. , 1987, The Journal of clinical investigation.

[53]  R. Rothlein,et al.  Reduction of central nervous system ischemic injury in rabbits using leukocyte adhesion antibody treatment. , 1991, Stroke.

[54]  S. Ben‐Sasson,et al.  Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation , 1992, The Journal of cell biology.

[55]  S. Chien,et al.  Quantitative studies on the influence of leukocytes on the vascular resistance in a skeletal muscle preparation. , 1984, Microvascular research.

[56]  W. Bursch,et al.  Cell death by apoptosis and its protective role against disease. , 1992, Trends in pharmacological sciences.

[57]  H. Steller.  Mechanisms and genes of cellular suicide , 1995, Science.

[58]  K. Mullane,et al.  Activated Neutrophils Release Mediators That May Contribute to Myocardial Injury and Dysfunction Associated with Ischemia and Reperfusion a , 1988, Annals of the New York Academy of Sciences.

[59]  M. Entman,et al.  The role of the neutrophil in myocardial ischemia and reperfusion. , 1996, EXS.

[60]  M. Chopp,et al.  Anti‐ICAM‐1 antibody reduces ischemic cell damage after transient middle cerebral artery occlusion in the rat , 1994, Neurology.

[61]  D. Vaux.  Toward an understanding of the molecular mechanisms of physiological cell death. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[62]  D. Priebat,et al.  Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. , 1982, The Journal of investigative dermatology.

[63]  Birdsall Hh.  Induction of ICAM-1 on human neural cells and mechanisms of neutrophil-mediated injury. , 1991 .

[64]  M. Chopp,et al.  Anti‐CD11b monoclonal antibody reduces ischemic cell damage after transient focal cerebral ischemia in rat , 1994, Annals of neurology.

[65]  C. Smith,et al.  Role of β1 β2 integrins and ICAM-1 in lung injury after deposition of IgG and IgA immune complexes , 1993 .

[66]  S. Werns,et al.  The role of the neutrophil and free radicals in ischemic myocardial injury. , 1989, Journal of molecular and cellular cardiology.

[67]  M. James,et al.  Conversion of the substrate specificity of mouse proteinase granzyme B , 1994, Nature Structural Biology.

[68]  J. Cohen,et al.  Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[69]  M. Ballestas,et al.  Regulation of intercellular adhesion molecule-1 gene expression by tumor necrosis factor-alpha, interleukin-1 beta, and interferon-gamma in astrocytes. , 1994, Journal of neuroimmunology.

[70]  M. Chopp,et al.  Anti-CD11b monoclonal antibody reduces ischemic cell damage after transient (2h) but not after permanent MCA occlusion in the rat , 1994 .

[71]  C. Purdie,et al.  Thymocyte apoptosis induced by p53-dependent and independent pathways , 1993, Nature.

[72]  P. Kochanek,et al.  Polymorphonuclear leukocyte accumulation in brain regions with low blood flow during the early postischemic period. , 1986, Stroke.

[73]  R. Rothlein,et al.  Reduction of central nervous system ischemic injury by monoclonal antibody to intercellular adhesion molecule. , 1991, Journal of neurosurgery.

[74]  M. Chopp,et al.  Temporal Profile of in situ DNA Fragmentation after Transient Middle Cerebral Artery Occlusion in the Rat , 1995, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[75]  J. Tschopp,et al.  Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins. , 1991, Biochemistry.

[76]  S. Korsmeyer,et al.  BCL2 protein is topographically restricted in tissues characterized by apoptotic cell death. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[77]  Michael Loran Dustin,et al.  Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families , 1988, Cell.

[78]  Z. Werb,et al.  Coordinated expression of extracellular matrix-degrading proteinases and their inhibitors regulates mammary epithelial function during involution , 1992, The Journal of cell biology.