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Granulocytes: The culprit in ischemic damage to the intestine
536
SELECTED
SUMMARIES
NEWER THERAPEUTIC STRATEGIES FOR GASTROINTESTINAL MALIGNANCIES Henderson GB, Tsuji JM [Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California) Methotrexate efflux in Ll210 cells. J Biol Chem 1987;262:13571-8. The chemotherapeutic agent methotrexate exits Ll210 mouse leukemia cells by multiple routes, including a unidirectional efflux component that is sensitive to bromosulfophthalein. In a series of experiments, these investigators further characterized the kinetic and specific properties of this latter efflux system. The contribution of the other efflux routes was eliminated by treatment of cells with an active ester of methotrexate and by reducing the assay pH to 6.2. The remaining efflux at this latter pH was >90% sensitive to bromosulfophthalein. A correlation was noted between (a) the sensitivity of this bromosulfophthalein-inhibitable methotrexate efflux system to inhibition by prostaglandin Al, probenecid, and certain metabolic inhibitors and (b) the ability of these same compounds to inhibit the unidirectional efflux of 3’,5’cyclic adenosine monophosphate in several cell lines, which suggests that this chemotherapeutic agent may share a common efflux route with cyclic nucleotides. Comment.
The majority of research involved in elucidating the specific actions of antineoplastic drugs on cancer cells has focused on deoxyribonucleic acid as a target for these agents. This concept has obviously proven productive, as it has led to the development of specific chemotherapeutic agents that have been useful for the treatment of cancer in humans. The insistence on deoxyribonucleic acid as the target for these drugs has, however. to some extent distracted the attention of investigators from other potential targets and mechanisms of these agents. Increasing attention has recently been focused on the concept that the plasma membranes of tumor cells may also represent a target for these drugs. There is now considerable evidence, for example, alkylating agents, antimethat at least 20 such drugs, including tabolites, and natural products such as adriamycin. bleomycin, and vinca alkaloids have distinct effects on the plasma membranes of various cancer cells (Muggia FM, ed. Experimental and clinical progress in cancer chemotherapy. Volume 4. The Hague: Martinus Nijhoff. 1985:81-131.). Based on these observations. it would appear that, at least in certain instances, the effects of these drugs on the surface membranes of tumor cells may represent the major mechanism(s) involved in their antineoplastic action. A large fraction of the published work in this area involves adriamycin and the anthracyclines. Adriamycin. perhaps secondary to its amphipathic structure, has been previously shown to have numerous effects on tumor cell membranes including actions on fusion, lectin interactions, ion gradients. lipid and protein composition, receptors, and lipid fluidity. (Muggia FM. ed. Experimental and clinical progress in cancer chemotherapy, Volume 4. The Hague: Martinus Nijhoff, 1985:81-131.). Based on the latter observations, it has been suggested that these effects, particularly those involving fluidity alterations, may be primary events in the initiation of the cytotoxicity of this agent, but further evidence to support these contentions is clearly needed. Cell surface membrane phenomena have also been suggested to be involved in anticancer drug resistance. For example, Glaubiger et al. (Proc Am Assoc Cancer Res 1983;284:284) have reported that P388 cells, resistant to adriamycin, were less fluid than sensitive cells. Along these same lines, examination of carbohydrate cells
GASTROENTEROLOGY
Vol. 95. No. 2
has revealed
the presence of a 170,000-dalton glycoprotein in the plasma membranes of mutants resistant to adriamycin. This protein, termed the “P-glycoprotein,” has been isolated and characterized (J Biol Chem 1979;254:12701-5). Similar glycoproteins have also been identified in the surface membranes of other tumor cells resistant to various chemotherapeutic agents (Cancer Res 1979;39:2070, 1983;43:222). It has been suggested that this family of glycoproteins may perhaps confer resistance to antineoplastic agents in these tumors by increasing the permeability of their plasma membranes, thereby leading to an increased efflux of these drugs. Taken together, these observations strongly suggest that the plasma membranes of tumor cells are intimately involved in the antineoplastic action of various chemotherapeutic agents. In this regard, the transport of methotrexate in and out of LIZ10 mouse leukemia cells has been extensively studied over the past few years. It would appear that the drug enters these cells via a single high-affinity, carrier-mediated system (J Biol Chem 1968;243:5007, 1984;259:1526). Methotrexate efflux. however, appears to be much more complex, involving at least three separate and distinct systems. The present studies by Henderson and Tsuji have now more extensively characterized one of these efflux routes. These studies potentially have a great deal of clinical relevance to the treatment of gastrointestinal tumors. It is obvious that our current chemotherapeutic approaches to almost all gastrointestinal malignancies need to be greatly modified, given response rates of -20% or less with various antineoplastic agents in patients with these cancers. In general, the vast majority of research in this area involves clinical trials using single or multiple poisons. The approach taken by Henderson and Tsuji. i.e., first elucidating the mechanism(s) involved in the action of Uluorouracil and other drugs on gastrointestinal tumor membranes and then rationally planning strategies based on this data. makes a great deal of sense and should be used in the area of gastrointestinal malignancies. Along these same lines, it has also become clear that the modifications of the lipid composition and physical state of the lipids of tumor cell membranes by dietary or other manipulations may have tremendous biological and therapeutic potential in this area [Cancer Res 1987:47:4529, Lipids 1987:22:178). In this regard, modification of the membrane properties of tumor membranes has already been shown to augment the cytotoxicity of various antineoplastic therapies, including thermal radiation and chemotherapy (Cancer Res 1987;47:4529). For example, tumor cells enriched with polyunsaturated fatty acids have been shown to have enhanced sensitivity to adriamytin, secondary to an increase in influx of this agent in these cells (Biochim Biophys Acta 1986;888:10). I strongly anticipate that, over the next several years, these and other novel approaches to the treatment of various cancers, including gastrointestinal malignancies, should lead to major advances in this area. T. A. BRASITLJS. M.D.
GRANULOCYTES: THE CULPRIT IN ISCHEMIC DAMAGE TO THE INTESTINE Hernandez LA, Grisham MB, Twohig B, Arfors KE, Harlan JM, Granger DN (Department of Physiology and Biophysics, Louisiana State University Medical Center, Shreveport, Louisiana). Role of neutrophils in ischemia-reperfusion-induced microvascular injury. Am J Physiol 1987; 253:H699-703. Injury to the intestine during ischemic events is caused at the time of reperfusion in part by increased production of the enzyme xanthine oxidase, which plays a central role
August
1988
in the formation of reactive oxygen metabolites. During &hernia-reperfusion injury, there is a massive increase in the rate of reactive oxygen metabolite production, despite the presence of naturally occurring degradative enzymes. It has been important to determine the nature of the cell types that contribute to the increase in reactive oxygen metabolites in ischemia-reperfusion injury to the intestine. The potential role of granulocytes in ischemia-reperfusion intestinal injury has been suggested by morphologic and pharmacologic approaches. In the present study, to examine neutrophils and their possible primary role in ischemia-reperfusion injury to the intestine, Hernandez and coworkers used two approaches: neutrophil depletion with polyclonal antiserum, and prevention of neutrophil adherence with a monoclonal antibody. In the animal model system used, local intestinal blood flow in cats was reduced to 15%-20% of control for 1 h, and the intestine was then reperfused, which results in highly reproducible ischemia-reperfusion intestinal injury. Hernandez and coworkers administered neutrophil antiserum, monoclonal antibodies to prevent neutrophil adherence, or control serum 1 h before the initiation of ischemia-reperfusion intestinal injury. The antiserum to cat neutrophils was demonstrated to lower the blood neutrophil count to ~5% of control values 1 h after treatment and resulted in neutrophil values that remained below 10% of control for the next 3 h. The monoclonal antibody 60.3 is directed against a specific membrane-associated glycoprotein (pchain of the CD18 complex) and was demonstrated not to cause any decrease in peripheral blood neutrophil count [the neutrophil count increased in the monoclonal antibody-treated animals]. Monoclonal antibody 60.3 caused a marked inhibition of neutrophil adherence that was doserelated and resulted in an 80% reduction of adherence of cat neutrophils in vitro. When either antineutrophil serum (used to reduce total granulocyte number) or monoclonal antibodies (used to prevent neutrophil adherence) were administered, a marked reduction in ischemia-reperfusion injury was observed. Ischemia-reperfusion injury in control experiments resulted in a marked increase in microvascular permeability of the intestine. This marked increase in microvascular permeability was attenuated by either neutrophil depletion or prevention of neutrophil adherence. In this interesting and important study, Hernandez and coworkers have demonstrated that neutrophil infiltration is a cause. rather than an effect, of ischemia-reperfusion-induced microvascular injury to the intestine. The intestinal mucosa is extremely sensitive to ischemia-induced damage. Enhanced mi-
Comment.
crovascular permeability is one of the early manifestations of ischemia-reperfusion injury. During ischemia and reperfusion, there is a marked increase in neutrophil migration into intestinal mucosa coupled with reduced glutathione and superoxide dismutase levels (Grisham et al., Am J Physiol 1986;251:G56774). Pretreatment with allopurinol (a xanthine-oxidase inhibitor) or superoxidase dismutase (an antioxidant enzyme) prevented the influx of neutrophiis and inhibited the decrease in glutathione levels (Grisham et at., Am J Physiol 1986;251:G567-74). These observations implicate granulocytes and oxygen radicals in ischemia-reperfusion-induced mucosal injury. The results of the present study by Hernandez and coworkers demonstrate that granulocvte depletion or prevention of granulocyte adherence to
SELECTED
SUMMARIES
537
the microvasculature will attenuate the microvascular permeability changes caused by ischemia-reperfusion. These observations support the concept that neutrophils accumulate in damaged mucosa in response to xanthine-oxidase activation and then become the primary mediator of the markedly enhanced oxygen radical-dependent injury produced during reperfusion of ischemic bowel. The study is also of interest with regard to pathophysiologic mechanisms in other inflammatory intestinal diseases, in particular ulcerative colitis and Crohn’s disease. 5-Aminosalicylic acid has been demonstrated to function as an oxygen radical scavenger 1985;16:377-84) and it is thus (Carlin et al., Agents Actions possible that one of the therapeutic mechanisms of action of 5-aminosalicylic acid in inflammatory bowel disease is related to inhibition of oxygen radical-mediated and granulocyte-mediated events. Because of its potent oxygen radical-scavenging capabilities, it would be of interest in future studies to examine the effects of 5aminosalicylic acid on ischemia-reperfusion injury to the intestine and radiation enteritis in animal model systems. The present study also provides a system to examine the sequence of events involved in neutrophil adherence to and chemotaxis across the microvascular endothelium. Definition of the nature of the chemotactic factors and cell surface molecules involved in controlling the migration of neutrophils into ischemic intestine could provide insight into events that are involved in intestinal inflammation I<. I’ MA, DEKMOTT. M.D.
GOOD NEWS AND BAD NEWS: PROSTAGLANDINS ALTER THE COURSE OF FULMINANT MURINE HEPATITIS BUT THE MICE DIE ANYWAY Abecassis M, Falk JA, Makowka L. et al. (Departments of Surgery and Medicine, University of Toronto, Toronto, Canada) 16,16 Dimethyl prostaglandin E2 prevents the development of fulminant hepatitis and blocks the induction of monocyteimacrophage procoagulant activity after murine hepatitis virus strain 3 infection. J Clin Invest 1987;80:881-9 (September). Certain prostaglandins have been shown to exert a hepatic cytoprotective effect in models of liver injury induced by hepatotoxic substances or hypoperfusion. In addition, specific prostaglandins appear to have both immunosuppressive and antiviral activity. The influence of 16,16-dimethyl prostaglandin E2 (dmPGEZ) on the fulminant hepatitis induced by murine hepatitis virus type 3 (MHV-3) was the focus of this study. Mice treated with dmPGE, before or up to 48 h after intraperitoneal inoculation with MHV-3 failed to develop the strikingly elevated serum alanine aminotransferase levels, hypoglycemia, metabolic acidosis, and confluent hepatic necrosis that were seen in mice infected with MHV-3 but untreated with dmPGE?. Delay of the initiation of dmPGE, treatment for 72 h failed to protect against hepatic necrosis. An analogue of dmPGE2. 16,16-dimethyl prostaglandin F,,, did not exert any protective effect. High titers of MHV-3 were recovered from liver homogenates of both untreated and dmPGE,-treated mice at 24, 48, 72. and 96 h after infection and the pathogenicity of the agent was not altered in the dmPGE,-treated animals. Splenic macrophages from MHV-3-infected animals demonstrated a dra-
SELECTED
SUMMARIES
NEWER THERAPEUTIC STRATEGIES FOR GASTROINTESTINAL MALIGNANCIES Henderson GB, Tsuji JM [Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California) Methotrexate efflux in Ll210 cells. J Biol Chem 1987;262:13571-8. The chemotherapeutic agent methotrexate exits Ll210 mouse leukemia cells by multiple routes, including a unidirectional efflux component that is sensitive to bromosulfophthalein. In a series of experiments, these investigators further characterized the kinetic and specific properties of this latter efflux system. The contribution of the other efflux routes was eliminated by treatment of cells with an active ester of methotrexate and by reducing the assay pH to 6.2. The remaining efflux at this latter pH was >90% sensitive to bromosulfophthalein. A correlation was noted between (a) the sensitivity of this bromosulfophthalein-inhibitable methotrexate efflux system to inhibition by prostaglandin Al, probenecid, and certain metabolic inhibitors and (b) the ability of these same compounds to inhibit the unidirectional efflux of 3’,5’cyclic adenosine monophosphate in several cell lines, which suggests that this chemotherapeutic agent may share a common efflux route with cyclic nucleotides. Comment.
The majority of research involved in elucidating the specific actions of antineoplastic drugs on cancer cells has focused on deoxyribonucleic acid as a target for these agents. This concept has obviously proven productive, as it has led to the development of specific chemotherapeutic agents that have been useful for the treatment of cancer in humans. The insistence on deoxyribonucleic acid as the target for these drugs has, however. to some extent distracted the attention of investigators from other potential targets and mechanisms of these agents. Increasing attention has recently been focused on the concept that the plasma membranes of tumor cells may also represent a target for these drugs. There is now considerable evidence, for example, alkylating agents, antimethat at least 20 such drugs, including tabolites, and natural products such as adriamycin. bleomycin, and vinca alkaloids have distinct effects on the plasma membranes of various cancer cells (Muggia FM, ed. Experimental and clinical progress in cancer chemotherapy. Volume 4. The Hague: Martinus Nijhoff. 1985:81-131.). Based on these observations. it would appear that, at least in certain instances, the effects of these drugs on the surface membranes of tumor cells may represent the major mechanism(s) involved in their antineoplastic action. A large fraction of the published work in this area involves adriamycin and the anthracyclines. Adriamycin. perhaps secondary to its amphipathic structure, has been previously shown to have numerous effects on tumor cell membranes including actions on fusion, lectin interactions, ion gradients. lipid and protein composition, receptors, and lipid fluidity. (Muggia FM. ed. Experimental and clinical progress in cancer chemotherapy, Volume 4. The Hague: Martinus Nijhoff, 1985:81-131.). Based on the latter observations, it has been suggested that these effects, particularly those involving fluidity alterations, may be primary events in the initiation of the cytotoxicity of this agent, but further evidence to support these contentions is clearly needed. Cell surface membrane phenomena have also been suggested to be involved in anticancer drug resistance. For example, Glaubiger et al. (Proc Am Assoc Cancer Res 1983;284:284) have reported that P388 cells, resistant to adriamycin, were less fluid than sensitive cells. Along these same lines, examination of carbohydrate cells
GASTROENTEROLOGY
Vol. 95. No. 2
has revealed
the presence of a 170,000-dalton glycoprotein in the plasma membranes of mutants resistant to adriamycin. This protein, termed the “P-glycoprotein,” has been isolated and characterized (J Biol Chem 1979;254:12701-5). Similar glycoproteins have also been identified in the surface membranes of other tumor cells resistant to various chemotherapeutic agents (Cancer Res 1979;39:2070, 1983;43:222). It has been suggested that this family of glycoproteins may perhaps confer resistance to antineoplastic agents in these tumors by increasing the permeability of their plasma membranes, thereby leading to an increased efflux of these drugs. Taken together, these observations strongly suggest that the plasma membranes of tumor cells are intimately involved in the antineoplastic action of various chemotherapeutic agents. In this regard, the transport of methotrexate in and out of LIZ10 mouse leukemia cells has been extensively studied over the past few years. It would appear that the drug enters these cells via a single high-affinity, carrier-mediated system (J Biol Chem 1968;243:5007, 1984;259:1526). Methotrexate efflux. however, appears to be much more complex, involving at least three separate and distinct systems. The present studies by Henderson and Tsuji have now more extensively characterized one of these efflux routes. These studies potentially have a great deal of clinical relevance to the treatment of gastrointestinal tumors. It is obvious that our current chemotherapeutic approaches to almost all gastrointestinal malignancies need to be greatly modified, given response rates of -20% or less with various antineoplastic agents in patients with these cancers. In general, the vast majority of research in this area involves clinical trials using single or multiple poisons. The approach taken by Henderson and Tsuji. i.e., first elucidating the mechanism(s) involved in the action of Uluorouracil and other drugs on gastrointestinal tumor membranes and then rationally planning strategies based on this data. makes a great deal of sense and should be used in the area of gastrointestinal malignancies. Along these same lines, it has also become clear that the modifications of the lipid composition and physical state of the lipids of tumor cell membranes by dietary or other manipulations may have tremendous biological and therapeutic potential in this area [Cancer Res 1987:47:4529, Lipids 1987:22:178). In this regard, modification of the membrane properties of tumor membranes has already been shown to augment the cytotoxicity of various antineoplastic therapies, including thermal radiation and chemotherapy (Cancer Res 1987;47:4529). For example, tumor cells enriched with polyunsaturated fatty acids have been shown to have enhanced sensitivity to adriamytin, secondary to an increase in influx of this agent in these cells (Biochim Biophys Acta 1986;888:10). I strongly anticipate that, over the next several years, these and other novel approaches to the treatment of various cancers, including gastrointestinal malignancies, should lead to major advances in this area. T. A. BRASITLJS. M.D.
GRANULOCYTES: THE CULPRIT IN ISCHEMIC DAMAGE TO THE INTESTINE Hernandez LA, Grisham MB, Twohig B, Arfors KE, Harlan JM, Granger DN (Department of Physiology and Biophysics, Louisiana State University Medical Center, Shreveport, Louisiana). Role of neutrophils in ischemia-reperfusion-induced microvascular injury. Am J Physiol 1987; 253:H699-703. Injury to the intestine during ischemic events is caused at the time of reperfusion in part by increased production of the enzyme xanthine oxidase, which plays a central role
August
1988
in the formation of reactive oxygen metabolites. During &hernia-reperfusion injury, there is a massive increase in the rate of reactive oxygen metabolite production, despite the presence of naturally occurring degradative enzymes. It has been important to determine the nature of the cell types that contribute to the increase in reactive oxygen metabolites in ischemia-reperfusion injury to the intestine. The potential role of granulocytes in ischemia-reperfusion intestinal injury has been suggested by morphologic and pharmacologic approaches. In the present study, to examine neutrophils and their possible primary role in ischemia-reperfusion injury to the intestine, Hernandez and coworkers used two approaches: neutrophil depletion with polyclonal antiserum, and prevention of neutrophil adherence with a monoclonal antibody. In the animal model system used, local intestinal blood flow in cats was reduced to 15%-20% of control for 1 h, and the intestine was then reperfused, which results in highly reproducible ischemia-reperfusion intestinal injury. Hernandez and coworkers administered neutrophil antiserum, monoclonal antibodies to prevent neutrophil adherence, or control serum 1 h before the initiation of ischemia-reperfusion intestinal injury. The antiserum to cat neutrophils was demonstrated to lower the blood neutrophil count to ~5% of control values 1 h after treatment and resulted in neutrophil values that remained below 10% of control for the next 3 h. The monoclonal antibody 60.3 is directed against a specific membrane-associated glycoprotein (pchain of the CD18 complex) and was demonstrated not to cause any decrease in peripheral blood neutrophil count [the neutrophil count increased in the monoclonal antibody-treated animals]. Monoclonal antibody 60.3 caused a marked inhibition of neutrophil adherence that was doserelated and resulted in an 80% reduction of adherence of cat neutrophils in vitro. When either antineutrophil serum (used to reduce total granulocyte number) or monoclonal antibodies (used to prevent neutrophil adherence) were administered, a marked reduction in ischemia-reperfusion injury was observed. Ischemia-reperfusion injury in control experiments resulted in a marked increase in microvascular permeability of the intestine. This marked increase in microvascular permeability was attenuated by either neutrophil depletion or prevention of neutrophil adherence. In this interesting and important study, Hernandez and coworkers have demonstrated that neutrophil infiltration is a cause. rather than an effect, of ischemia-reperfusion-induced microvascular injury to the intestine. The intestinal mucosa is extremely sensitive to ischemia-induced damage. Enhanced mi-
Comment.
crovascular permeability is one of the early manifestations of ischemia-reperfusion injury. During ischemia and reperfusion, there is a marked increase in neutrophil migration into intestinal mucosa coupled with reduced glutathione and superoxide dismutase levels (Grisham et al., Am J Physiol 1986;251:G56774). Pretreatment with allopurinol (a xanthine-oxidase inhibitor) or superoxidase dismutase (an antioxidant enzyme) prevented the influx of neutrophiis and inhibited the decrease in glutathione levels (Grisham et at., Am J Physiol 1986;251:G567-74). These observations implicate granulocytes and oxygen radicals in ischemia-reperfusion-induced mucosal injury. The results of the present study by Hernandez and coworkers demonstrate that granulocvte depletion or prevention of granulocyte adherence to
SELECTED
SUMMARIES
537
the microvasculature will attenuate the microvascular permeability changes caused by ischemia-reperfusion. These observations support the concept that neutrophils accumulate in damaged mucosa in response to xanthine-oxidase activation and then become the primary mediator of the markedly enhanced oxygen radical-dependent injury produced during reperfusion of ischemic bowel. The study is also of interest with regard to pathophysiologic mechanisms in other inflammatory intestinal diseases, in particular ulcerative colitis and Crohn’s disease. 5-Aminosalicylic acid has been demonstrated to function as an oxygen radical scavenger 1985;16:377-84) and it is thus (Carlin et al., Agents Actions possible that one of the therapeutic mechanisms of action of 5-aminosalicylic acid in inflammatory bowel disease is related to inhibition of oxygen radical-mediated and granulocyte-mediated events. Because of its potent oxygen radical-scavenging capabilities, it would be of interest in future studies to examine the effects of 5aminosalicylic acid on ischemia-reperfusion injury to the intestine and radiation enteritis in animal model systems. The present study also provides a system to examine the sequence of events involved in neutrophil adherence to and chemotaxis across the microvascular endothelium. Definition of the nature of the chemotactic factors and cell surface molecules involved in controlling the migration of neutrophils into ischemic intestine could provide insight into events that are involved in intestinal inflammation I<. I’ MA, DEKMOTT. M.D.
GOOD NEWS AND BAD NEWS: PROSTAGLANDINS ALTER THE COURSE OF FULMINANT MURINE HEPATITIS BUT THE MICE DIE ANYWAY Abecassis M, Falk JA, Makowka L. et al. (Departments of Surgery and Medicine, University of Toronto, Toronto, Canada) 16,16 Dimethyl prostaglandin E2 prevents the development of fulminant hepatitis and blocks the induction of monocyteimacrophage procoagulant activity after murine hepatitis virus strain 3 infection. J Clin Invest 1987;80:881-9 (September). Certain prostaglandins have been shown to exert a hepatic cytoprotective effect in models of liver injury induced by hepatotoxic substances or hypoperfusion. In addition, specific prostaglandins appear to have both immunosuppressive and antiviral activity. The influence of 16,16-dimethyl prostaglandin E2 (dmPGEZ) on the fulminant hepatitis induced by murine hepatitis virus type 3 (MHV-3) was the focus of this study. Mice treated with dmPGE, before or up to 48 h after intraperitoneal inoculation with MHV-3 failed to develop the strikingly elevated serum alanine aminotransferase levels, hypoglycemia, metabolic acidosis, and confluent hepatic necrosis that were seen in mice infected with MHV-3 but untreated with dmPGE?. Delay of the initiation of dmPGE, treatment for 72 h failed to protect against hepatic necrosis. An analogue of dmPGE2. 16,16-dimethyl prostaglandin F,,, did not exert any protective effect. High titers of MHV-3 were recovered from liver homogenates of both untreated and dmPGE,-treated mice at 24, 48, 72. and 96 h after infection and the pathogenicity of the agent was not altered in the dmPGE,-treated animals. Splenic macrophages from MHV-3-infected animals demonstrated a dra-