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Lactoferrin and monocyte biology
Inflammation
15.1
INFLAMMATION, OXYGEN RADICALS AND TISSUE INJURY Peter A. Ward Department of Pathology, University of Michigan School of Medicine, Ann Arbor, MI 48109, U.S.A. Oxygen products generated either from activated phagocytic cells or xanthine oxidase (X.O.) have been shown to participate in the generation of chemotactic factors for neutrophils, in vivo studies involving immune complex-induced injury suggest that O2" (perhaps derived from X.O.) but not H202 is involved in neutrophil recruitment. Although deposition of immune complexes in lung and in dermis results in acute tissue injury that is complement and neutrophil-dependent, only in the lung does the injury appear to be related to the role of toxic oxygen products from neutrophils. These findings suggest fundamentally different mechanisms of PMN-mediated injury of endothelial cells in the two vascular beds. On the basis of in vitro studies, rat pulmonary artery endothelial cells show evidence of synergistic interaction between H202 and leukocytic proteases, resulting in amplification of endothelial cell injury. The targets of H202 and the proteases and the sequence of events are not known. Finally, damage of endothelial cells by activated PMN is associated with conversion of intracellular xanthine dehydrogenase (X.D.) to X.O. via mechanism that is independent of the role of oxygen products. Evidence that 02" generation is important in the outcome of endothelial cell damage following contact with either activated neutrophils or H202 is shown by the protective effects of X.O. inhibitors (allopurinol, oxypurinol) or by loading of endothelial cells with superoxide dismutase. These findings suggest a complexity in ways in which oxygen radicals bring about injury during the inflammatory response.
15.3
EX VIVO MONITOR OF SUPEROXIDE G~NERATION BY INVADED NEUTROPHILS IN P~RI-MYOCARDITIS Hiroe Nakazawa, Yoshinori Saigusa, Kenryo K Minezaki, Kohji Ichimori, Hamlk~ Okino, T ~ h i k o Murata, Nobuhiko 0hnlshi, Ikuko Ueno Tokal University, Isehara, 259-11, Japan Previous studies on superoxide (02.) generation of neutrophils (PMN) have utilized circulating PMN in vitro by which functions of tissue PMN can not be evaluated. We established a method which enables observation of 0 2. from epicardial PMN in ex vivo. Using rats, peri-myocarditis was produced by inoculating salmonella (S) in a pericardial space. After 8 or 24 hours of inoculation (8-H and 24-H groups), hearts were removed, retro-perfused with Langendorff mode and placed upside down in a dark box with a photon counter to avoid effluent chemiluminescence (Che-L). Luciferin analog which is specific for 0 2. was used as a Che-L probe. Hearts without S exhibited basic Che-L of only 363 counts/sec (cps) without responding to phorbol myristate acetate (PMA) stimulation since PMN were not accumulated. In 8-H group, hqsic Che-L was low but rose to 8169 cps after PMA stimulation. In contrast, 24-H group showed a markedly elevated basic Che-L (5354 cps) prior to PMA stimulation. Azlministration of superoxide dismutase completely eliminated Che-L of basic and peak levels. Histological e~mlnation disclosed that numerous PMN were accumulated on the eplcardium in beth groups. This is the first report showing spontaneous 02. generation from tissue PMN and this method elucidated that PMN were not activated at an earlier inf1~mmatory stage but began to produce 0 2. without exogenous stimulatio~ at a later stage.
THE RESPIRATORY BURST OXIDASE OF HUMAN NEUTROPHILS Bernard M. Babior Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, CA 92037, U.S.A.
139
15.2
The r e s p i r a t o r y burst is a metabolic pathway whose purpose is the production of microbicidal oxidants by the partial reduction of oxygen. This pathway occurs in activated phagocytes and (at much lower levels) in Blymphocytes. The reactive oxidants produced via this pathway participate in microbial killing, but are also t h o u g h t to be i n v o l v e d in the pathogenesis of such disorders as the acute respiratory distress syndrome and reperfusion injury. The key enzyme of the respiratory burst is the respiratory burst oxidase, a highly regulated enzyme that catalyzes the r e d u c t i o n of o x y g e n to O 2- at the e x p e n s e of NADPH. The respiratory burst oxidase is attached to the m e m b r a n e c y t o s k e l e t o n , p r o b a b l y to c o n t r o l the geographic distribution of O~- production in the activated phagocyte. The enzyme is a complex flavohemoprotein containing at least 5 different types of subunits. In the resting neutrophil these subunits are distributed between the p l a s m a m e m b r a n e and t h e c y t o s o l , but u p o n activation of the cell the cytosolic subunits move to the plasma membrane to assemble the active oxidase. Usually the oxidase continues to produce O~- until it is killed by o x i d a t i o n , but u n d e r some c i r c u m s t a n c e s it can be returned to the resting state, to be reactivated when the cell is stimulated again.
LACTOFERRIN AND MONOCYTE BIOLOGY Myron S. Cohen, Jonathan S. Serody, Larry Charniga, Department of Medicine, University of North Carolina, Chapel Hill, N.C. 27599, USA We have demonstrated that human apolactoferrin is capable of limiting formation of hydroxyl radical generated by stimulated neutrophils (Biochem J. 264:447, 1989), or by monocytic phagocytes (J. Exp. Med 168:2367, 1989). In the case of monocytic phagocytes, which do not make apolactoferrin, we find this compound rapidly ingested by receptor mediated endocytosis. Ingested lactoferrin limits HO" formation by cells stimulated with phorbol in the presence of Fe +3 (Clin Res. 38:351A, 198~). Monocyte derived macrophages express 3.36x i0 receptors per cell with a Kd of 3.56 x i0- M. Undifferentiated monocytoid U937 ce~is express receptors with a Kd of 6.87 x 10-JM. When incubated with 500ug of lactoferrin U937 cells concentrated 150 ug of this protein after I hour. U937 cells were used to isolate the lactoferrin receptor by affinity chromatography. Using cell membranes a 27 Kd protein which retained the ability to bind apolactoferrin was isolated. Using a micelle preparation a larger Ii0 Kd protein was obtained. We believe the 27 Kd protein represents a lactoferrin binding domain. Monocytes which have ingested apolactoferrin are limited in their ability to generate HO', which could enhance their survival and influence the inflammatory response.
15.4
15.1
INFLAMMATION, OXYGEN RADICALS AND TISSUE INJURY Peter A. Ward Department of Pathology, University of Michigan School of Medicine, Ann Arbor, MI 48109, U.S.A. Oxygen products generated either from activated phagocytic cells or xanthine oxidase (X.O.) have been shown to participate in the generation of chemotactic factors for neutrophils, in vivo studies involving immune complex-induced injury suggest that O2" (perhaps derived from X.O.) but not H202 is involved in neutrophil recruitment. Although deposition of immune complexes in lung and in dermis results in acute tissue injury that is complement and neutrophil-dependent, only in the lung does the injury appear to be related to the role of toxic oxygen products from neutrophils. These findings suggest fundamentally different mechanisms of PMN-mediated injury of endothelial cells in the two vascular beds. On the basis of in vitro studies, rat pulmonary artery endothelial cells show evidence of synergistic interaction between H202 and leukocytic proteases, resulting in amplification of endothelial cell injury. The targets of H202 and the proteases and the sequence of events are not known. Finally, damage of endothelial cells by activated PMN is associated with conversion of intracellular xanthine dehydrogenase (X.D.) to X.O. via mechanism that is independent of the role of oxygen products. Evidence that 02" generation is important in the outcome of endothelial cell damage following contact with either activated neutrophils or H202 is shown by the protective effects of X.O. inhibitors (allopurinol, oxypurinol) or by loading of endothelial cells with superoxide dismutase. These findings suggest a complexity in ways in which oxygen radicals bring about injury during the inflammatory response.
15.3
EX VIVO MONITOR OF SUPEROXIDE G~NERATION BY INVADED NEUTROPHILS IN P~RI-MYOCARDITIS Hiroe Nakazawa, Yoshinori Saigusa, Kenryo K Minezaki, Kohji Ichimori, Hamlk~ Okino, T ~ h i k o Murata, Nobuhiko 0hnlshi, Ikuko Ueno Tokal University, Isehara, 259-11, Japan Previous studies on superoxide (02.) generation of neutrophils (PMN) have utilized circulating PMN in vitro by which functions of tissue PMN can not be evaluated. We established a method which enables observation of 0 2. from epicardial PMN in ex vivo. Using rats, peri-myocarditis was produced by inoculating salmonella (S) in a pericardial space. After 8 or 24 hours of inoculation (8-H and 24-H groups), hearts were removed, retro-perfused with Langendorff mode and placed upside down in a dark box with a photon counter to avoid effluent chemiluminescence (Che-L). Luciferin analog which is specific for 0 2. was used as a Che-L probe. Hearts without S exhibited basic Che-L of only 363 counts/sec (cps) without responding to phorbol myristate acetate (PMA) stimulation since PMN were not accumulated. In 8-H group, hqsic Che-L was low but rose to 8169 cps after PMA stimulation. In contrast, 24-H group showed a markedly elevated basic Che-L (5354 cps) prior to PMA stimulation. Azlministration of superoxide dismutase completely eliminated Che-L of basic and peak levels. Histological e~mlnation disclosed that numerous PMN were accumulated on the eplcardium in beth groups. This is the first report showing spontaneous 02. generation from tissue PMN and this method elucidated that PMN were not activated at an earlier inf1~mmatory stage but began to produce 0 2. without exogenous stimulatio~ at a later stage.
THE RESPIRATORY BURST OXIDASE OF HUMAN NEUTROPHILS Bernard M. Babior Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, CA 92037, U.S.A.
139
15.2
The r e s p i r a t o r y burst is a metabolic pathway whose purpose is the production of microbicidal oxidants by the partial reduction of oxygen. This pathway occurs in activated phagocytes and (at much lower levels) in Blymphocytes. The reactive oxidants produced via this pathway participate in microbial killing, but are also t h o u g h t to be i n v o l v e d in the pathogenesis of such disorders as the acute respiratory distress syndrome and reperfusion injury. The key enzyme of the respiratory burst is the respiratory burst oxidase, a highly regulated enzyme that catalyzes the r e d u c t i o n of o x y g e n to O 2- at the e x p e n s e of NADPH. The respiratory burst oxidase is attached to the m e m b r a n e c y t o s k e l e t o n , p r o b a b l y to c o n t r o l the geographic distribution of O~- production in the activated phagocyte. The enzyme is a complex flavohemoprotein containing at least 5 different types of subunits. In the resting neutrophil these subunits are distributed between the p l a s m a m e m b r a n e and t h e c y t o s o l , but u p o n activation of the cell the cytosolic subunits move to the plasma membrane to assemble the active oxidase. Usually the oxidase continues to produce O~- until it is killed by o x i d a t i o n , but u n d e r some c i r c u m s t a n c e s it can be returned to the resting state, to be reactivated when the cell is stimulated again.
LACTOFERRIN AND MONOCYTE BIOLOGY Myron S. Cohen, Jonathan S. Serody, Larry Charniga, Department of Medicine, University of North Carolina, Chapel Hill, N.C. 27599, USA We have demonstrated that human apolactoferrin is capable of limiting formation of hydroxyl radical generated by stimulated neutrophils (Biochem J. 264:447, 1989), or by monocytic phagocytes (J. Exp. Med 168:2367, 1989). In the case of monocytic phagocytes, which do not make apolactoferrin, we find this compound rapidly ingested by receptor mediated endocytosis. Ingested lactoferrin limits HO" formation by cells stimulated with phorbol in the presence of Fe +3 (Clin Res. 38:351A, 198~). Monocyte derived macrophages express 3.36x i0 receptors per cell with a Kd of 3.56 x i0- M. Undifferentiated monocytoid U937 ce~is express receptors with a Kd of 6.87 x 10-JM. When incubated with 500ug of lactoferrin U937 cells concentrated 150 ug of this protein after I hour. U937 cells were used to isolate the lactoferrin receptor by affinity chromatography. Using cell membranes a 27 Kd protein which retained the ability to bind apolactoferrin was isolated. Using a micelle preparation a larger Ii0 Kd protein was obtained. We believe the 27 Kd protein represents a lactoferrin binding domain. Monocytes which have ingested apolactoferrin are limited in their ability to generate HO', which could enhance their survival and influence the inflammatory response.
15.4