Role of leukocyte CDIUCDI8 complex in endotoxic and septic shock in rabbits JOHN R. THOMAS, JOHN M. HARLAN, CHARLES L. RICE, AND Departments of Anesthesiology, Medicine, Surgery, and Physiology-Biophysics, University of Washington, Seattle, Washington 98195 THOMAS, JOHN R., JOHN M. HARLAN, CHARLES L. RICE, AND ROBERT K. WINN. Role of leukocyte CD1 1 /CD1 8 complex in endotoxic and septic shock in rabbits. J. Appl. Physiol. 73(4): 1510-1516, 1992.-Two models of sepsis were investigated using rabbits. In the first model, rabbits given lipopolysaccharide (LPS) were treated with saline (group II) or CD18 monoclonal antibody (MAb) 60.3 (group III). Group I animals received no LPS. Cardiac output was maintained by infusion of lactated Ringer solution with group II (95 t 68 ml/kg) requiring significantly more than group I (0 * 0 ml/kg) or group III (39 * 27 ml/kg). Lung permeability indexes in groups II (median 0.002, range 0.023) and III (median 0.0035, range 0.053) were not different but were significantly greater than group I (median 0.0007, range 0.001). In the second model, peritonitis was produced by devascularizing the appendix, leaving it in situ for 19 h, and then performing an appendectomy. Saline or MAb 60.3 treatment was at appendectomy and every 12 h for 3 days. Survival was significantly greater in the MAb 60.3-treated group at day IO (90 vs. 40%). Lung permeability was increased at day 2 and was not different between groups. Day 1 fluid requirements were greater in the saline-treated group. These data are consistent with MAb 60.3 protection of systemic but not pulmonary circulation in two models of sepsis. sepsis; adult respiratory distress syndrome; multiple organ failure; leukocyte adhesion molecules; integrins; neutrophil-mediated injury; monoclonal antibody; lung injury; systemic injury
(MOFS)isasignificant cause of death in trauma and surgical patients requiring intensive care, and it is the leading cause of delayed death in these patients (10). The initiating event is thought to be an increased permeability of blood vessels resulting in edema formation, tissue anoxia, cell death, and subsequent organ failure. MOFS appears to result from generalized inflammation characterized by fever, leukocytosis, generalized vasodilation, generalized edema, and failure of multiple organ systems. The organs that fail include lung, liver, kidney, cardiovascular system, central nervous system, and alimentary tract (9,10). Sepsis or sepsis syndrome is thought to be one of the leading causes of this generalized inflammation (8, 9). Neutrophils (PMNs) have been shown to cause endothelial cell injury either in vivo or in vitro, but they must be tightly adherent to produce the injury. That is, circulating anti-inflammatory molecules are in such excess under normal conditions that toxic products produced by PMNs are quickly neutralized. PMN adherence creates a MULTIPLEORGANFAILURESYNDROME
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protected local microenvironment between PMNs and endothelial cells where the concentration of PMN toxins can overcome circulating anti-inflammatory agents (27, 28). This microenvironment also restricts entrance of additional circulating anti-inflammatory agents. Alternatively, PMN-PMN aggregation can cause tissue injury if aggregation is sufficient to cause vascular occlusion with downstream ischemia and subsequent tissue death. Either of these two pathways can cause vascular injury leading to organ damage and organ failure as seen in MOFS. Leukocytes (presumably PMNs) caused organ injury and late death in both monkeys and rabbits following hemorrhagic shock (21,30,31). These same cells have been implicated in organ injury associated with septic shock. Also, neutrophils were implicated in the pathogenesis of endotoxin-induced acute lung injury in dogs (32, 33) and sheep (13) and in the human disease adult respiratory distress syndrome (ARDS) (14). However, there is experimental and clinical evidence to suggest that PMNs are not necessary for acute lung injury. PMN depletion did not consistently prevent endotoxin-induced lung injury in goats (34) and monkeys (25), and ARDS has been reported to occur in neutropenic patients (16, 18, 23). One of the primary mechanisms of PMN adherence and aggregation involves binding of the CD1 1/CD18 glycoprotein complex to its counterstructure on endothelial cells (11). Monoclonal antibody (MAb) 60.3 inhibits PMN adherence and aggregation by binding to a functional epitope on CD18 (1). The function of MAb 60.3 is specific for adherence in that other PMN functions such as granule release and oxidant production in response to soluble stimuli are not affected (5). We investigated the hypothesis that PMNs produce adverse physiological effects via CD1 1/CDl%dependent adherence and/or aggregation in septic shock. In experiments described in this paper, we examined the effect of MAb 60.3 administration to rabbits under two experimental conditions. In one set of experiments animals were given an intravenous infusion of lipopolysaccharide (LPS), and in a second set animals were made septic by devascularizing their appendix. METHODS
Animals. New Zealand White rabbits were used in these experiments. This study adhered to the National Institutes of Health “Guidelines for the Use of Labora-
0 1992 the American
Physiological
Society
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tory Animals,” and the protocols were approved by the University of Washington Animal Care Committee. Animalpreparation (LPS infusion). Animals were anesthetized via a marginal ear vein with ketamine and supplemental local lidocaine. A 3-Fr double-lumen thermistor-tipped catheter was placed in the aorta via the common carotid artery, and a 3.5Fr catheter was placed in the vena cava via the jugular vein. The aortic catheter was used to monitor pressure, sense cold for cardiac output determinations, and obtain serial blood samples for blood gas determinations. The venous catheter was used to inject drugs, resuscitative fluids, and cold saline for cardiac output determinations. Rabbits were allowed a l-h stabilization period before the experiment was begun. Experimental protocol (LPS infusion). Animals were randomly assigned to the saline and MAb 60.3 treatment groups with the investigator unaware of the assignment. However, no attempt was made to prevent the investigator from knowing which animals were in the control (no LPS) group. The treatment groups were given MAb 60.3 (2 mg/kg) or saline (equal volume). Five minutes after treatment, they received an intravenous infusion of Escherichia coli LPS (3 pglkg) in 10 ml saline over 30 min. The animals breathed room air throughout the experiment. Thermal dilution cardiac output and aortic pressure were measured every 30 min. Attempts were made to maintained cardiac output within 10% of baseline by infusion of lactated Ringer solution. Baseline cardiac output was determined by averaging the values obtained during the 1 h between catheter placement and treatment. Leukocyte count, hematocrit, blood gases, and pH were determined from 0.5 ml of blood aspirated hourly from the arterial catheter. Five hours after treatment the animals were killed with an overdose of ketamine for determination of lung vascular protein permeability and gravimetric lung water calculations. Animal preparation (sepsis). Rabbits were divided into two experimental protocols in these sepsis experiments. They were anesthetized and sepsis was induced by devascularizing the appendix and ligating it at the base. All animals were given lactated Ringer solution (50 ml) as fluid replacement after closure of the laporatomy. This was followed by a second laporatomy 19 h later to remove the necrotic appendix. Animals in the first protocol were given lactated Ringer solution for 3 days after the second laporatomy to prevent dehydration. Fluid replacement consisted of lactated Ringer solution in a volume equal to twice their weight loss on day 1 and equal to their weight loss on days 2 and 3. The person responsible for care of the animals was unaware of the treatment protocol. Animals in the first group were randomly divided into two groups of 10 each and treated with the antibiotic cefazolin (20 mg/kg) every 12 h for 3 days. One-half of these rabbits were treated with the MAb 60.3 and onehalf with saline. The loading dose of MAb 60.3 was 2 mg/kg, and this was followed by 1 mg/kg every 12 h for 3 days to ensure saturation of CDl8. These animals were followed for 10 days. Animals that lost ~20% of their baseline body weight were assumed to be irreversibly septic and were killed. A previous study in our laboratory using a similar experimental preparation showed that
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~3% of septic rabbits survived a weight loss of ~20%. Animals with excessive weight loss were thus killed for humanitarian reasons. At the end of 10 days, KaplanMeier survival curves were constructed, and the difference between groups was evaluated by log-rank test. The second group of animals were made septic by appendiceal devascularization, ligation, and removal at 19 h as described above. In addition, catheters were placed into the vena cava vi .a jugula .r vein and into the aorta via carotid artery at the time of appendectomy as described in the LPS experiments. Rabbits were divided into two groups and given either saline or MAb 60.3 in addition to antibiotics (cefazolin, 20 mg/kg). These animals were then monitored for 6 h after appendectomy before they were returned to their cages. Lactated Ringer solution was used for resuscitation fluid and was given to maintain cardiac output at -250 ml/kg (normal cardiac output in rabbits). On the following day they were returned to the laboratory where monitoring and resuscitation were continued for an additional 6 h, and then the rabbits were killed. Lung permeability was measured postmortem as described below. Cardiac output determinations. Cardiac output was determined by thermodilution using a l-ml saline injectate at O°C into the central venous catheter. Temperature and thermodilution cardiac output calculations were made using a cardiac output computer. Cardiac outputs are expressed in milliliters per kilogram per minute to correct for differences in body weight among animals. The percent of baseline values for cardiac output were calculated and used for statistical analysis. Blood cell counts. Hematocrits were measured by capillary microcentrifugation. Leukocyte counts were performed by hand using a hemocytometer. Blood gas, pH, and bicarbonate determinations. PO,, Pco,, and pH of whole blood at 37°C were determined using a pH/blood gas analyzer. The Henderson-Hasselbath equation was used to calculate bicarbonate concentration. Pulmonary vascular protein permeability. One hour before the end of the study animals were given an intravenous injection of 1251-labeled bovine serum albumin (-2.5 &i). Immediately after death the chest was opened, the right hilum was clamped, and the righ .t lung was removed for gravimetric extravascular lung water determination (EVLW; see below). The trachea was intubated, and the left lung was gently lavaged in and out four times with 20 ml of saline. Ten milliliters of blood were withdrawn from the heart into a heparinized SYringe. Samples of lavage fluid, blood, and homogenized right lung were weighed, and then radioactivity was measured in a gamma scintillation counter. Plasma 1251-albumin was calculated from blood concentration and hematocrit because all 1251-albumin was extracellular [i.e., plasma concentration = blood concentration/( 1 - hematocrit)]. Lung permeability index was defined as the ratio of lavage to plasma concentration of 1251-albumin. A second indicator of lung permeability is the ratio of 1251-albumin in EVLW to plasma. We determined the total 1251albumin in EVLW (corrected for vascular volume) and calculated the ratio of that value to plasma normalized to body weight.
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1. Cardiac output values. Cardiac output was maintained within 10% of baseline in animals treated with monoclonal antibody (MAb) 60.3 by infusion of lactated Ringer solution. Administration of lactated Ringer solution could not maintain cardiac output within 10% of baseline in saline-treated animals. FIG.
Gravimetric lung water determinations. The right lung was weighed and homogenized with a volume of water equal to the lung weight. Weighed samples of lung homogenate and blood were placed in an oven at -7OOC for 1 wk to determine dry weights. A sample of the lung homogenate was freeze-thawed and centrifuged at 35,000 g for 1 h, and then hemoglobin concentrations were measured from the supernatant. EVLW was calculated by the method of Pearce et al. (24) and normalized to body weight (BW). Statistics. Data are expressed as either means t SD or median and range. Data in the figures are shown as mean t SE unless otherwise noted. Analysis of variance (ANOVA) for repeated measures was used to test for significance; then the F test was used to test which groups were different (2). The Kruskal-Wallis H test (29) was used to test for significance between lung permeability indexes, and the Wilcoxon rank test was used to test for significance between fluid requirements of septic animals. These data did not have a normal distribution; thus nonparametric statistics were used. The Wilcoxon rank sum test (26) was used after the Kruskal-Wallis H test to determine which data were different from each other, and the conservative Bonferroni adjustment was employed to avoid the problem of multiple sampling. Differences were assumed to be significant at P < 0.05. A4Ab 60.3. MAb 60.3 is a murine immunoglobulin G,, and was aseptically collected from mouse ascites and then purified as previously described (1). The purified MAb solution was tested for LPS by limulus assay and by the whole blood tumor necrosis factor production assay (4) and found to have no detectable LPS contamination. The whole blood assay is capable of detecting as little as 0.01 rig/ml of LPS. RESULTS
LPS infusion. Two animals from the saline-treated group and two from the MAb 60.3-treated group died from cardiovascular failure before the study ended. One of the MAb 60.3-treated animals that died received 228 ml/kg of lactacted Ringer before death, which is more
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than 8 SDS above the mean (see below) for the MAb 60.3 group. This animal was considered an outlier and was excluded from statistical analysis and from all figures. The reason for this excessive fluid requirement is unknown. Lung vascular protein permeability and EVLW could not be determined for those animals that died prematurely. Hemodynamics (LPS). Cardiac output decreased in all animals after infusion of LPS. Infusion of lactated Ringer solution, however, allowed cardiac output to be maintained to within 10% of baseline value in animals given MAb 60.3 but not in those given saline (Fig. 1). Cardiac output was not significantly different between control and MAb 60.3-treated animals; however, average cardiac output of the saline group was significantly different from both the control and MAb 60.3 groups (ANOVA and F test). Aortic blood pressure did not change significantly over time and did not differ significantly between groups. Fluid requirements (LPS). The cumulative lactated Ringer requirements are displayed in Fig. 2. Control animals (i.e., animals not given LPS) did not need fluid resuscitation. The average lactated Ringer requirement was 39 t 27 ml/kg for the MAb 60.3-treated group and 95 + 68 ml/kg for the saline-treated group. The final fluid requirements for these groups were significantly different by ANOVA. The saline-treated group was different from the other two groups, but the MAb 60.3 and control group were not different by the Scheffe’s F test. White blood cell counts (LPS). The average leukocyte counts from all three groups are shown in Fig. 3. The average number of circulating leukocytes in control animals differed significantly from the animals given LPS. There was a leukocytosis resulting from the placement of catheters in this group; whereas leukopenia resulted in the two groups receiving LPS. The leukocyte counts did not differ significantly between saline- and MAb 60.3treated animals. Hematocrit averaged 36 t 1.9,36 t 2.1, and 35 t 1.1 at baseline for MAb 60.3-treated, salinetreated, and control groups, respectively. Averages of hematocrit at 5 h were 32 t 2.1, 29 t 2.4, and 34 t 2.9 for MAb 60.3-treated, saline-treated, and control animals, respectively. The hematocrit of animals given MAb 60.3 -
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FIG. 2. Cumulative fluid requirements. Saline group received significantly more fluid than MAb 60.3 or control group. Fluid requirements were not significantly different between control and MAb 60.3 groups.
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Time 3. Circulating leukocyte counts. kocytes associated with lipopolysaccharide by MAb 60.3 treatment. FIG.
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FIG. 4. Survival curves for rabbits and ligating their appendix. One-half saline and one-half with MAb 60.3.
30
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rabbits. Animals of appendectomy
Time (hours) in circulating leuwas not prevented
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did not differ significantly from either of the other two groups; however, the control and saline-treated animals were significantly different when hematocrits were compared by ANOVA. Blood gases, pH, and bicarbonate (LPS). There were no differences in arterial pH (-7.4) or arterial oxygen tension (m 75 Torr) between groups, and they did not change with time. Calculated arterial bicarbonate concentration was 18.8 t 3.217.7 t 2.8, and 19.2 t 2.4 at baseline in the MAb 60.3-treated, saline-treated, and control animals, respectively. It was 12.2 t 1.9,11.3 t 2.5, and 19.2 t 3.5 at 5 h in the MAb 60.3-treated, saline-treated, and control animals, respectively. PCO, was 30 t 4.9, 29 t 4.3, and 30 t 4.0 Torr at baseline in MAb 60.3-treated, salinetreated, and control animals, respectively. It was 16.5 t 2.4, 17.3 t 3.1, and 26.8 t 4.2 Torr at 5 h for MAb 60.3treated, saline-treated, and control animals, respectively. Bicarbonate and PCO, were significantly different between groups by ANOVA for repeated measures. The MAb 60.3-treated and saline-treated groups were not significantly different for these two variables; however, both of these groups were different from control by ANOVA for repeated measures. Survivors (sepsis). Survival curves for the 10 days of
5. Cumulative fluid requirements were treated with either saline or MAb and every 12 h for 3 days. FIG.
this experiment are shown in Fig. 4 for the saline- and MAb 60.3-treated animals. There was a 10% mortality in the MAb 60.3-treated group and a 60% mortality in the saline-treated group. The one death in the MAb 60.3treated group occurred between days 4 and 5. Four of the control animals were killed because of excessive weight loss on days 3, 4, 7, and 9. The two animals that died of their sepsis died on days 2 and 9. The two groups were statistically different by log-rank test (P < 0.05). Fluid requirements (sepsis). Fluid requirements for the septic animals are shown in Fig. 5. It is important to note that both groups of animals had necrotic appendexes The first carwhen the appendectomy was performed. disc o UtP ut was measured within 30 min of the appendectomy. The lactated Ringer solution required by the saline-treated animals began to diverge from the MAb 60.3-treated animals within the first few hours of obserperiod vation. At the end of the first 6-h observation these animals required a median of 36.7 with a range 128.7 ml/kg, whereas the MAb 60.3-treated animals required only a median of 7.0 with a range of 35.7 ml/kg. At the end of the experiment the saline-treated animals had received a median of 64.3 with a range of 133.6 ml/kg, whereas the MAb 60.3-treated animals had received median of 44.5 with a range of 64.9 ml/kg. The difference in fluid requirements between these two groups of animals was statistically significant at the end of the first day by Wilcoxon rank-sum test (P < 0.05). Significance was lost by the second day following free access to food and water overnight. Cardiac output and systemic pressure (sepsis). There was no statistical difference in the cardiovascular function in these two groups of animals as measured by cardiac output and aortic pressure. Lung vascular permeability and E VL W (LPS). EVLW I BW ratio of the right lung did not differ between groups at 2.33 t 0.25, 2.29 t 0.39, and 2.07 t 0.35 for the MAb 60.3-treated, saline-treated, and control groups, respectively. In addition there were no statistical differences between EVLW-to-plasma-to-BW 1251-albumin ratios between any of the groups. The median lavage-to-plasma 1251-albumin ratio was 0.0035 with a range of 0.053 for the
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a range of 0.016 in the MAb 60.3-treated animals and to 0.0041 with a range of 0.0057 in the saline-treated animals. Arterial oxygerzation (sepsis). Arterial PO, decreased in both the saline- and MAb 60.3-treated septic groups. The decrease with time was significant by ANOVA; however, there was no difference between the two groups. In the saline-treated group, PO, decreased from 89.9 t 7.6 to 68 + 10.7 Torr, and in the MAb 60.3-treated group it decreased from 94.2 t 16.0 to 72.0 t 2.9 Torr at -24 h. Arterial PCO, and pH did not change significantly between the initial and final measurement. DISCUSSION
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FIG. 6. Lung permeability index measured as ratio of 1251-labeled albumin in lavage fluid to 1251- albumi n in plasm a. Permeability indexes for animals given lipopolysaccharide (A) and for animals made septic (B) are shown. Lavage was performed 5 h after administration of lipopolysaccharide or 48 h after appendectomy in MAb 60.3- and salinetreated animals. Normal group received no treatment.
MAb 60.3 group, 0.002 with a range of 0.023 for the saline group, and 0.0007 with a range of 0.001 for the control group. The lavage-to-plasma 1251-albuminratio for all of these animals is shown in Fig. 6A. The three groups were significantly different when compared by the KruskalWallace H test, and the control group was different from both the MAb 60.3- and the saline-treated groups. There was no difference between the two groups given LPS. Permeability index (sepS;s).The ratio of 1251-albuminin bronchoalveolar lavage fluid to that in plasma for the two groups in this study together with normal controls is shown in Fig. 6B. This permeability index was significantly increased above normal in both the saline- and MAb 60.3-treated animals compared with the normal controls. The medial value for normal animals was 0.0007 with a range of 0.001 and increased to 0.0014 with
The CD18 MAb 60.3 allowed us to investigate the role of one pathway of neutrophil adherence and/or aggregation after LPS infusion in rabbits. There was a reduction in the resuscitative fluid requirement after LPS infusion in animals given MAb 60.3. The septic rabbits in the saline-treated group also required more fluid than the MAb 60.3-treated on the 1st day of sepsis. Because all septic animals had free accessto food and water overnight, it is not realistic to compare these animals after the 1st day. Survival was significantly increased at day 10 in the septic animals treated with MAb 60.3 compared with the saline-treated group. These results are consistent with neutrophils causing significant adverse effects as a result of sepsis or after endotoxin infusion and with the injury being CDlUCDl8 dependent. MAb 60.3 provided no protection to the lungs in these experiments as permeability of the lungs was not different between saline- and MAb 60.3-treated animals (either septic animals or after LPS infusion). Thus the protection provided by MAb 60.3 relative to decreased fluid requirements and increased survival suggestsprotection of some systemic organs. Cardiac output could not be maintained after LPS infusion in the saline-treated animals even with increased fluid administration. These results suggest that CDllI CDl8-dependent neutrophil injury may occur to the myocardium during endotoxic shock. Although other factors could explain these results (e.g., decreased preload), evidence of myocardial injury has been reported. Transvenous endomyocardial biopsies revealed intravascular stasis of neutrophils, focal neutrophil infiltration, and occasional myocyte necrosis by light microscopy in dogs given intraperitoneal Pseudomonas aeruginosas. Endothelial swelling and interstitial edema were seen by electron microscopy (22). The metabolic acidosis after infusion of LPS can be assumed to result from increased lactic acid production. Neither serum lactate nor electrolyte concentration (for ion-gap calculations) was measured in these experiments. This presumed lactic acidosis may have resulted from excess tissue production as a result of hypoperfusion or inadequate oxygen extraction or from diminished hepatic clearance of lactate. Determination of the cause of acidosis was beyond the scope of this study. From our results it would appear that neutrophil adherence and/or aggregation through a CDll/CDl8 pathway was not in-
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volved in establishing the metabolic acidosis associated with endotoxic shock. Neutropenia after infusion of LPS and other stimuli is a common finding. In two separate studies the number of circulating radiolabeled neutrophils declined after intravenous LPS administration, and the neutrophils were shown to be sequestered in the pulmonary vascular bed (3,lZ). The experiments described here showed that the leukopenia seen after LPS infusion was not CDll/CD18 dependent because there was no difference between leukocyte counts in the saline-treated and MAb 60.3-treated rabbits. These findings are consistent with those seen by Lundberg and Wright (17) and Doerschuk et al. (6) after infusion of zymosan-activated serum or chemotactic peptide. Decreases in PMN deformability after activation with zy mosan-ac tivated plasma was demonstrated in vitro and proposed as a mechanism of neutropenia in vivo (15). This could account for the leukopenia we observed. These experi ments suggest that the endotheli al damage induced by perito nitis or LPS infusion was ameliorated by the CD18 MAb 60.3 in the systemic but not the pulmonary circulation. That is, permeability index increased in both septic animals and animals given LPS without regard to treatment with MAb 60.3. In addition, PO, decreased in the septic animals and was not different between saline and MAb 60.3 treatment. This suggests that adherence in the lung may result from a pathway that is CD18independent. Such a pathway has been characterized by Doerschuk et al. (7) in the lung and by Mileski et al. (19) in a macrophage-enriched peritoneum. Doerschuk et al. showed that neutrophil emigration in response to Streptococcus pneumoniae bacteria and hydrochloric acid instillation into the lung was CD18 independent. In the same study emigration toward the same stimuli in the systemic circulation was CD18 dependent. Mileski et al. showed that the mechanism of emigration into the peritoneum was dependent on whether macrophages were present. Macrophage enrichment converted the emigration mechanism from CD18 dependent to partially CD‘18 independent. Sepsis is the greatest single risk factor for development of ARDS in critically ill patients, and PMNs have been implicated as causing this syndrome. It is thought that the pulmonary edema seen in ARDS is the result of increased permeability in the lungs. In the present study sepsis caused an increase in the ratio of 12”1-albumin in bronchoalveolar lavage to 1251-albumin in plasma. This is consistent with an increase in permeability. Also, arterial PO, decreased with time in the septic animals, a finding consistent with respiratory distress. There was no difference in permeability index or arterial oxygenation in the animals treated with saline or MAb 60.3 after infusion of LPS. In a previous study from our laboratory we examined safety aspects of MAb 60.3 in abdominal sepsis by pretreating rabbits with MAb 60.3 at the time of devascularization and again at appendectomy (20). In that study we found equivalent mortality at 10 days and equivalent infectious complications (defined as abdominal abscess formation and/or wound infections) in saline- and MAb 60.3-treated animals. The present study was designed to
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examine the effectiveness of MAb 60.3 treatment of established abdominal sepsis. Both groups of animals in the present study were allowed to develop sepsis over 19 h and then divided into two groups for treatment with either saline or MAb 60.3. During development of sepsis, PMNs and monocytes were able to respond normally to the septic challenge by emigrating to the peritoneum in their host defense role. However, the cycle of inflammation was broken at 19 h with MAb 60.3 with an apparent reduction in the vascular injury. In summary, we examined the role of neutrophil adherence and aggregation in sepsis and after LPS infusion using CD18 MAb 60.3. Our findings suggest that neutrophils cause a physiological derangement during sepsis and endotoxemia and that MAb 60.3 therapy may prevent the cardiovascular dysfunction associated with these conditions. Also, CD18 MAb 60.3 improved survival in sepsis when given over an extended period. The authors thank W. Purcell and R. Knake-Othberg for superb technical assistance. The MAb 60.3 was a gracious gift from Dr. Patrick Beatty, Fred Hutchinson Cancer Center, Seattle, WA. This study was supported by National Heart, Lung, and Blood Institute Grants HL-43141 and HL-30542 and by a grant from the American Heart Association. Address for reprint requests: R. K. Winn, Dept. of Surgery, Harborview Medical Center (ZA-16), 325 Ninth Ave., Seattle, WA 98104. Received
9 December
1991; accepted
in final
form
1 May
1992.
REFERENCES 1. BEATTY, P. G., J. A. LEDBETTER, P. J. MARTIN, T. H. PRICE, AND J. A. HANSEN. Definition of a common leukocyte cell-surface antigen (Lp95 150) associated with diverse cell-mediated immune functions. J. Immunol. 131: 2913-2918, 1983. 2. BRUNING, J. L., AND B. L. KINTZ. ComputationaL Handbook of Statistics. Glenview, IL: Scott, Foresman, 1977. 3. CYBULSKY, M. I., AND H. Z. MOVAT. Experimental bacterial pneumonia in rabbits: polymorphonuclear leukocyte margination and sequestration in rabbit lungs and quantitation and kinetics of ‘%rlabeled polymorphonuclear leukocytes in E. coli-induced lung lesions. Exp. Lung Res. 4: 47-66, 1982. 4. DESCH, C., N. KOVACH, W. PRESENT, C. BROYLES, AND J. HARLAN. Production of human tumor necrosis factor from whole blood ex vivo. Lymphokine Res. 8: 141-146, 1989. 5. DIENER, A. M., P. G. BEATTY, H. D. OCHS, AND J. M. HARLAN. The role of neutrophil membrane glycoprotein 150 (GP-150) in neutroPhil-mediated endothelial cell injury in vitro. J. Immunol. 135: 537543, 1985. 6. DOERSCHUK, C. M., D. ENGLISH, J. M. HARLAN, AND J. C. HOGG. The role of CD18 in neutropenia and neutrophil (PMN) sequestration induced by infusion of activated plasma (Abstract). FASEB J. 4: 496A, 1990. 7. DOERSCHUK, C. M., R. K. WINN, H. 0. COXSON, AND J. M. HARLAN. CD18-dependent and -independent mechanisms of neutrophi1 emigration in the pulmonary and systemic microcirculation of rabbits. J. Immunol. 144: 2327-2333, 1990. 8. FAIST, E., A. E. BAUE, H. DITTMER, AND G. HEBERER. Multiple organ failure in polytrauma patients. J. Trauma 23: 775-787, 1983. 9. FRY, D. E., L. PEARLSTEIN, R. L. FULTON, AND H. C. POLK. Multiple system organ failure: the role of uncontrolled infection. Arch. Surg. 115: 136-140, 1980. 10. GORIS, R. J. A., T. P. TE BOEKHORST, J. K. S. NUYTINCK, AND J. S. F. GIMBRERE. Multiple organ failure: generalized autodestructive inflammation? Arch. Surg. 120: 1109-1115, 1985. 11. HARLAN, J. M., B. R. SCHWARTZ, W. J. WALLIS, AND T. H. POHLMAN. The role of neutrophil membrane proteins in neutrophil emigration. In: Leukocyte Emigration and Its Sequelae, edited by H. Z. Movat. Krager: Basel, 1987, p. 94-104. 12. HASLETT, C., G. S. WORTHEN, P. C. GICLAS, D. C. MORRISON, J. E. HENSON, AND P. M. HENSON. The pulmonary vascular sequestra-
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14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
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tion of neutrophils in endotoxemia is initiated by an effect of endotoxin on the neutrophil in the rabbit. Am. Reu. Respir. Dis. 136: 9-181987. HEFLIN, A. C., AND K. L. BRIGHAM. Prevention by granulocyte depletion of increased vascular permeability of sheep lung following endotoxemia. J. Clin. Invest. 68: 1253-1260, 1981. HYERS, T. M., AND A. A. FOWLER. Adult respiratory distress syndrome: causes, morbidity, and mortality. Federation Proc. 45: 2529, 1986. INANO, H., AND C. M. DOERSCHUK. The effect of zymosan-activated plasma on neutrophil (PMN) deformability in vitro (Abstract). Am. Rev. Respir. Dis. 143: 329A, 1991. LAUFE, M. D., R. H. SIMON, A. FLINT, AND J. F. KELLER. Adult respiratory distress syndrome in neutropenic patients. Am. J. Med. 80:1022-1026,1986. LUNDBERG, C., AND S. D. WRIGHT. Relation of the CDll/CD18 family of leukocyte antigens to the transient neutropenia caused by chemoattractants. Blood 76: 1240-1245, 1990. MAUNDER, R. J., R. C. HACKMAN, E. RIFF, R. K. ALBERT, AND S. S. SPRINGMEYER. Occurrence of the adult respiratory distress syndrome in neutropenic patients. Am. Rev. Respir. Dis. 133: 313-316, 1986. MILESKI, W., J. HARLAN, C. RICE, AND R. WINN. Streptococcus pneumoniae-stimulated macrophages induce neutrophils to emigrate by a CD18-independent mechanism of adherence. Circ. Shock 31: 259-267,199O. MILESKI, W. J., R. K. WINN, J. M. HARLAN, AND C. L. RICE. Transient inhibition of neutrophil adherence with the anti-CD18 monoclonal antibody 60.3 does not increase mortality rates in abdominal sepsis. Surgery 109: 497-501, 1991. MILESKI, W. J., R. K. WINN, N. V. VEDDER, T. H. POHLMAN, J. M. HARLAN, AND C. L. RICE. Inhibition of CD18-dependent neutrophil adherence reduces organ injury after hemorrhagic shock in primates. Surgery 108: 205-212, 1990. NATANSON, C., R. E. CUNNION, D. A. BARRETT, K. W. PEART, R. L. DANNER, J. J. CONKLIN, T. J. MACVITTIE, R. I. WALKER, V. J. FERRANS, AND J. E. PARRILLO. Reversible myocardial dysfunction in a canine model of septic shock is associated with myocardial microcirculatory damage and focal neutrophil infiltration (Abstract). Clin. Res. 34: A639, 1986. OGNIBENE, F. P., S. E. MARTIN, M. M. PARKER, T. SCHLESINGER,
NEUTROPHIL
24. 25.
26.
27.
28.
29. 30.
31.
32.
33.
34.
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P. ROACH, C. BURCH, J. H. SELHAMER, AND J. E. PARRILLO. Adult respiratory distress syndrome in patients with severe neutropenia. N. Engl. J. Med. 315: 547-551, 1986. PEARCE, M. L., J. YAMASHITA, AND J. BEAZELL. Measurement of pulmonary edema. Circ. Res. 41: 482-488, 1965. PINGLETON, W. W., J. J. COALSON, AND C. A. GUENTER. Significance of leukocytes in endotoxic shock. Exp. Mol. Puthol. 22: 183194, 1975. REMINGTON, R. D. AND M. A. SCHORK. Statistics With Applications to the Biological and Health Sciences. Englewood Cliffs, NJ: Prentice Hall, 1970. SACKS, T., C. F. MOLDOW, P. R. CRADDOCK, T. K. BOWERS, AND H. S. JACOB. Oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes. J. Clin. Invest. 61: 11611167, 1978. SHASBY, D. M., S. S. SHASBY, AND M. J. PEACH. Granulocytes and phorbol myristate acetate increase permeability to albumin of cultured endothelial monolayers and isolated perfused lungs. Role of oxygen radicals and granulocytes. Am. Rev. Respir. Dis. 127: 72-76, 1983. SPRINGTHALL, R. C. Basic Statistical Analysis. Reading, MA: Addison-Wesley, 1982. VEDDER, N. B., B. W. FOUTY, R. K. WINN, J. M. HARLAN, AND C. L. RICE. Role of neutrophils in generalized reperfusion injury associated with resuscitation from shock. Surgery 106: 509-516, 1989. VEDDER, N. B., R. K. WINN, C. L. RICE, E. CHI, K.-E. ARFORS, AND J. M. HARLAN. A monoclonal antibody to the adherence promoting leukocyte glycoprotein CD18 reduces organ injury and improves survival from hemorrhagic shock and resuscitation in rabbits. J. Clin. Invest. 81: 939-944, 1988. WELSH, C. H., D. C. LIEN, G. S. WORTHEN, P. M. HENSON, AND J. V. WEIL. Endotoxin-pretreated neutrophils increase vascular permeability in dogs. J. Appl. Physiol. 66: 112-119, 1989. WELSH, C. H., D. C. LIEN, G. S. WORTHEN, AND J. V. WEIL. Pentoxifylline decreases endotoxin-induced pulmonary neutrophil sequestration and extravascular protein accumulation in the dog. Am. Rev. Respir. Dis. 138: 1106-1114, 1988. WINN, R., R. MAUNDER, E. CHI, AND J. HARLAN. Neutrophil depletion does not prevent lung edema after endotoxin infusion in goats. J. Appl. Physiol. 62: 116-121, 1987.
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(MOFS)isasignificant cause of death in trauma and surgical patients requiring intensive care, and it is the leading cause of delayed death in these patients (10). The initiating event is thought to be an increased permeability of blood vessels resulting in edema formation, tissue anoxia, cell death, and subsequent organ failure. MOFS appears to result from generalized inflammation characterized by fever, leukocytosis, generalized vasodilation, generalized edema, and failure of multiple organ systems. The organs that fail include lung, liver, kidney, cardiovascular system, central nervous system, and alimentary tract (9,10). Sepsis or sepsis syndrome is thought to be one of the leading causes of this generalized inflammation (8, 9). Neutrophils (PMNs) have been shown to cause endothelial cell injury either in vivo or in vitro, but they must be tightly adherent to produce the injury. That is, circulating anti-inflammatory molecules are in such excess under normal conditions that toxic products produced by PMNs are quickly neutralized. PMN adherence creates a MULTIPLEORGANFAILURESYNDROME
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protected local microenvironment between PMNs and endothelial cells where the concentration of PMN toxins can overcome circulating anti-inflammatory agents (27, 28). This microenvironment also restricts entrance of additional circulating anti-inflammatory agents. Alternatively, PMN-PMN aggregation can cause tissue injury if aggregation is sufficient to cause vascular occlusion with downstream ischemia and subsequent tissue death. Either of these two pathways can cause vascular injury leading to organ damage and organ failure as seen in MOFS. Leukocytes (presumably PMNs) caused organ injury and late death in both monkeys and rabbits following hemorrhagic shock (21,30,31). These same cells have been implicated in organ injury associated with septic shock. Also, neutrophils were implicated in the pathogenesis of endotoxin-induced acute lung injury in dogs (32, 33) and sheep (13) and in the human disease adult respiratory distress syndrome (ARDS) (14). However, there is experimental and clinical evidence to suggest that PMNs are not necessary for acute lung injury. PMN depletion did not consistently prevent endotoxin-induced lung injury in goats (34) and monkeys (25), and ARDS has been reported to occur in neutropenic patients (16, 18, 23). One of the primary mechanisms of PMN adherence and aggregation involves binding of the CD1 1/CD18 glycoprotein complex to its counterstructure on endothelial cells (11). Monoclonal antibody (MAb) 60.3 inhibits PMN adherence and aggregation by binding to a functional epitope on CD18 (1). The function of MAb 60.3 is specific for adherence in that other PMN functions such as granule release and oxidant production in response to soluble stimuli are not affected (5). We investigated the hypothesis that PMNs produce adverse physiological effects via CD1 1/CDl%dependent adherence and/or aggregation in septic shock. In experiments described in this paper, we examined the effect of MAb 60.3 administration to rabbits under two experimental conditions. In one set of experiments animals were given an intravenous infusion of lipopolysaccharide (LPS), and in a second set animals were made septic by devascularizing their appendix. METHODS
Animals. New Zealand White rabbits were used in these experiments. This study adhered to the National Institutes of Health “Guidelines for the Use of Labora-
0 1992 the American
Physiological
Society
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tory Animals,” and the protocols were approved by the University of Washington Animal Care Committee. Animalpreparation (LPS infusion). Animals were anesthetized via a marginal ear vein with ketamine and supplemental local lidocaine. A 3-Fr double-lumen thermistor-tipped catheter was placed in the aorta via the common carotid artery, and a 3.5Fr catheter was placed in the vena cava via the jugular vein. The aortic catheter was used to monitor pressure, sense cold for cardiac output determinations, and obtain serial blood samples for blood gas determinations. The venous catheter was used to inject drugs, resuscitative fluids, and cold saline for cardiac output determinations. Rabbits were allowed a l-h stabilization period before the experiment was begun. Experimental protocol (LPS infusion). Animals were randomly assigned to the saline and MAb 60.3 treatment groups with the investigator unaware of the assignment. However, no attempt was made to prevent the investigator from knowing which animals were in the control (no LPS) group. The treatment groups were given MAb 60.3 (2 mg/kg) or saline (equal volume). Five minutes after treatment, they received an intravenous infusion of Escherichia coli LPS (3 pglkg) in 10 ml saline over 30 min. The animals breathed room air throughout the experiment. Thermal dilution cardiac output and aortic pressure were measured every 30 min. Attempts were made to maintained cardiac output within 10% of baseline by infusion of lactated Ringer solution. Baseline cardiac output was determined by averaging the values obtained during the 1 h between catheter placement and treatment. Leukocyte count, hematocrit, blood gases, and pH were determined from 0.5 ml of blood aspirated hourly from the arterial catheter. Five hours after treatment the animals were killed with an overdose of ketamine for determination of lung vascular protein permeability and gravimetric lung water calculations. Animal preparation (sepsis). Rabbits were divided into two experimental protocols in these sepsis experiments. They were anesthetized and sepsis was induced by devascularizing the appendix and ligating it at the base. All animals were given lactated Ringer solution (50 ml) as fluid replacement after closure of the laporatomy. This was followed by a second laporatomy 19 h later to remove the necrotic appendix. Animals in the first protocol were given lactated Ringer solution for 3 days after the second laporatomy to prevent dehydration. Fluid replacement consisted of lactated Ringer solution in a volume equal to twice their weight loss on day 1 and equal to their weight loss on days 2 and 3. The person responsible for care of the animals was unaware of the treatment protocol. Animals in the first group were randomly divided into two groups of 10 each and treated with the antibiotic cefazolin (20 mg/kg) every 12 h for 3 days. One-half of these rabbits were treated with the MAb 60.3 and onehalf with saline. The loading dose of MAb 60.3 was 2 mg/kg, and this was followed by 1 mg/kg every 12 h for 3 days to ensure saturation of CDl8. These animals were followed for 10 days. Animals that lost ~20% of their baseline body weight were assumed to be irreversibly septic and were killed. A previous study in our laboratory using a similar experimental preparation showed that
NEUTROPHIL
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~3% of septic rabbits survived a weight loss of ~20%. Animals with excessive weight loss were thus killed for humanitarian reasons. At the end of 10 days, KaplanMeier survival curves were constructed, and the difference between groups was evaluated by log-rank test. The second group of animals were made septic by appendiceal devascularization, ligation, and removal at 19 h as described above. In addition, catheters were placed into the vena cava vi .a jugula .r vein and into the aorta via carotid artery at the time of appendectomy as described in the LPS experiments. Rabbits were divided into two groups and given either saline or MAb 60.3 in addition to antibiotics (cefazolin, 20 mg/kg). These animals were then monitored for 6 h after appendectomy before they were returned to their cages. Lactated Ringer solution was used for resuscitation fluid and was given to maintain cardiac output at -250 ml/kg (normal cardiac output in rabbits). On the following day they were returned to the laboratory where monitoring and resuscitation were continued for an additional 6 h, and then the rabbits were killed. Lung permeability was measured postmortem as described below. Cardiac output determinations. Cardiac output was determined by thermodilution using a l-ml saline injectate at O°C into the central venous catheter. Temperature and thermodilution cardiac output calculations were made using a cardiac output computer. Cardiac outputs are expressed in milliliters per kilogram per minute to correct for differences in body weight among animals. The percent of baseline values for cardiac output were calculated and used for statistical analysis. Blood cell counts. Hematocrits were measured by capillary microcentrifugation. Leukocyte counts were performed by hand using a hemocytometer. Blood gas, pH, and bicarbonate determinations. PO,, Pco,, and pH of whole blood at 37°C were determined using a pH/blood gas analyzer. The Henderson-Hasselbath equation was used to calculate bicarbonate concentration. Pulmonary vascular protein permeability. One hour before the end of the study animals were given an intravenous injection of 1251-labeled bovine serum albumin (-2.5 &i). Immediately after death the chest was opened, the right hilum was clamped, and the righ .t lung was removed for gravimetric extravascular lung water determination (EVLW; see below). The trachea was intubated, and the left lung was gently lavaged in and out four times with 20 ml of saline. Ten milliliters of blood were withdrawn from the heart into a heparinized SYringe. Samples of lavage fluid, blood, and homogenized right lung were weighed, and then radioactivity was measured in a gamma scintillation counter. Plasma 1251-albumin was calculated from blood concentration and hematocrit because all 1251-albumin was extracellular [i.e., plasma concentration = blood concentration/( 1 - hematocrit)]. Lung permeability index was defined as the ratio of lavage to plasma concentration of 1251-albumin. A second indicator of lung permeability is the ratio of 1251-albumin in EVLW to plasma. We determined the total 1251albumin in EVLW (corrected for vascular volume) and calculated the ratio of that value to plasma normalized to body weight.
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INHIBITION z c
125
3 9, ‘5 0 0
50-
.? 2
25-
9
0
OF
CD18-DEPENDENT
1
-
Saline (kll) CD18 MAb 60.3 (n=9) Control (kg)
-
0
I 1
-
1 2
Time
-
1 3
-
, 4
.
1
5
(hours)
1. Cardiac output values. Cardiac output was maintained within 10% of baseline in animals treated with monoclonal antibody (MAb) 60.3 by infusion of lactated Ringer solution. Administration of lactated Ringer solution could not maintain cardiac output within 10% of baseline in saline-treated animals. FIG.
Gravimetric lung water determinations. The right lung was weighed and homogenized with a volume of water equal to the lung weight. Weighed samples of lung homogenate and blood were placed in an oven at -7OOC for 1 wk to determine dry weights. A sample of the lung homogenate was freeze-thawed and centrifuged at 35,000 g for 1 h, and then hemoglobin concentrations were measured from the supernatant. EVLW was calculated by the method of Pearce et al. (24) and normalized to body weight (BW). Statistics. Data are expressed as either means t SD or median and range. Data in the figures are shown as mean t SE unless otherwise noted. Analysis of variance (ANOVA) for repeated measures was used to test for significance; then the F test was used to test which groups were different (2). The Kruskal-Wallis H test (29) was used to test for significance between lung permeability indexes, and the Wilcoxon rank test was used to test for significance between fluid requirements of septic animals. These data did not have a normal distribution; thus nonparametric statistics were used. The Wilcoxon rank sum test (26) was used after the Kruskal-Wallis H test to determine which data were different from each other, and the conservative Bonferroni adjustment was employed to avoid the problem of multiple sampling. Differences were assumed to be significant at P < 0.05. A4Ab 60.3. MAb 60.3 is a murine immunoglobulin G,, and was aseptically collected from mouse ascites and then purified as previously described (1). The purified MAb solution was tested for LPS by limulus assay and by the whole blood tumor necrosis factor production assay (4) and found to have no detectable LPS contamination. The whole blood assay is capable of detecting as little as 0.01 rig/ml of LPS. RESULTS
LPS infusion. Two animals from the saline-treated group and two from the MAb 60.3-treated group died from cardiovascular failure before the study ended. One of the MAb 60.3-treated animals that died received 228 ml/kg of lactacted Ringer before death, which is more
NEUTROPHIL
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than 8 SDS above the mean (see below) for the MAb 60.3 group. This animal was considered an outlier and was excluded from statistical analysis and from all figures. The reason for this excessive fluid requirement is unknown. Lung vascular protein permeability and EVLW could not be determined for those animals that died prematurely. Hemodynamics (LPS). Cardiac output decreased in all animals after infusion of LPS. Infusion of lactated Ringer solution, however, allowed cardiac output to be maintained to within 10% of baseline value in animals given MAb 60.3 but not in those given saline (Fig. 1). Cardiac output was not significantly different between control and MAb 60.3-treated animals; however, average cardiac output of the saline group was significantly different from both the control and MAb 60.3 groups (ANOVA and F test). Aortic blood pressure did not change significantly over time and did not differ significantly between groups. Fluid requirements (LPS). The cumulative lactated Ringer requirements are displayed in Fig. 2. Control animals (i.e., animals not given LPS) did not need fluid resuscitation. The average lactated Ringer requirement was 39 t 27 ml/kg for the MAb 60.3-treated group and 95 + 68 ml/kg for the saline-treated group. The final fluid requirements for these groups were significantly different by ANOVA. The saline-treated group was different from the other two groups, but the MAb 60.3 and control group were not different by the Scheffe’s F test. White blood cell counts (LPS). The average leukocyte counts from all three groups are shown in Fig. 3. The average number of circulating leukocytes in control animals differed significantly from the animals given LPS. There was a leukocytosis resulting from the placement of catheters in this group; whereas leukopenia resulted in the two groups receiving LPS. The leukocyte counts did not differ significantly between saline- and MAb 60.3treated animals. Hematocrit averaged 36 t 1.9,36 t 2.1, and 35 t 1.1 at baseline for MAb 60.3-treated, salinetreated, and control groups, respectively. Averages of hematocrit at 5 h were 32 t 2.1, 29 t 2.4, and 34 t 2.9 for MAb 60.3-treated, saline-treated, and control animals, respectively. The hematocrit of animals given MAb 60.3 -
120 Gi -5 100 -
E
J
saline (kll) CD 18 MAb 60.3 (n=9) Control (kg)
T
T
80
-201 0
1
2
Time
3
4
5
(hours)
FIG. 2. Cumulative fluid requirements. Saline group received significantly more fluid than MAb 60.3 or control group. Fluid requirements were not significantly different between control and MAb 60.3 groups.
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INHIBITION
= 10000 =t 3=
-
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CD18-DEPENDENT
NEUTROPHIL
ADHERENCE
z x
Saline (kll) CD 18 Mab 60.3 (n=9) Control (kg)
y
lOO-
IN
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Saline (n = 8) CD18MAb60.3(n=6)
l
8000
8 ; 6000 aI s 0 4000 s 3 a3 2000 4 1
2
3
Time 3. Circulating leukocyte counts. kocytes associated with lipopolysaccharide by MAb 60.3 treatment. FIG.
4
5
Reduction infusion
c 06
CD18 MAb 60.3 (n = 10) Saline (n q 10)
FIG. 4. Survival curves for rabbits and ligating their appendix. One-half saline and one-half with MAb 60.3.
30
in septic 60.3 at time
rabbits. Animals of appendectomy
Time (hours) in circulating leuwas not prevented
8
Time
25
5
(hours) ,
(days) made septic by devascularizing of the animals were treated with
did not differ significantly from either of the other two groups; however, the control and saline-treated animals were significantly different when hematocrits were compared by ANOVA. Blood gases, pH, and bicarbonate (LPS). There were no differences in arterial pH (-7.4) or arterial oxygen tension (m 75 Torr) between groups, and they did not change with time. Calculated arterial bicarbonate concentration was 18.8 t 3.217.7 t 2.8, and 19.2 t 2.4 at baseline in the MAb 60.3-treated, saline-treated, and control animals, respectively. It was 12.2 t 1.9,11.3 t 2.5, and 19.2 t 3.5 at 5 h in the MAb 60.3-treated, saline-treated, and control animals, respectively. PCO, was 30 t 4.9, 29 t 4.3, and 30 t 4.0 Torr at baseline in MAb 60.3-treated, salinetreated, and control animals, respectively. It was 16.5 t 2.4, 17.3 t 3.1, and 26.8 t 4.2 Torr at 5 h for MAb 60.3treated, saline-treated, and control animals, respectively. Bicarbonate and PCO, were significantly different between groups by ANOVA for repeated measures. The MAb 60.3-treated and saline-treated groups were not significantly different for these two variables; however, both of these groups were different from control by ANOVA for repeated measures. Survivors (sepsis). Survival curves for the 10 days of
5. Cumulative fluid requirements were treated with either saline or MAb and every 12 h for 3 days. FIG.
this experiment are shown in Fig. 4 for the saline- and MAb 60.3-treated animals. There was a 10% mortality in the MAb 60.3-treated group and a 60% mortality in the saline-treated group. The one death in the MAb 60.3treated group occurred between days 4 and 5. Four of the control animals were killed because of excessive weight loss on days 3, 4, 7, and 9. The two animals that died of their sepsis died on days 2 and 9. The two groups were statistically different by log-rank test (P < 0.05). Fluid requirements (sepsis). Fluid requirements for the septic animals are shown in Fig. 5. It is important to note that both groups of animals had necrotic appendexes The first carwhen the appendectomy was performed. disc o UtP ut was measured within 30 min of the appendectomy. The lactated Ringer solution required by the saline-treated animals began to diverge from the MAb 60.3-treated animals within the first few hours of obserperiod vation. At the end of the first 6-h observation these animals required a median of 36.7 with a range 128.7 ml/kg, whereas the MAb 60.3-treated animals required only a median of 7.0 with a range of 35.7 ml/kg. At the end of the experiment the saline-treated animals had received a median of 64.3 with a range of 133.6 ml/kg, whereas the MAb 60.3-treated animals had received median of 44.5 with a range of 64.9 ml/kg. The difference in fluid requirements between these two groups of animals was statistically significant at the end of the first day by Wilcoxon rank-sum test (P < 0.05). Significance was lost by the second day following free access to food and water overnight. Cardiac output and systemic pressure (sepsis). There was no statistical difference in the cardiovascular function in these two groups of animals as measured by cardiac output and aortic pressure. Lung vascular permeability and E VL W (LPS). EVLW I BW ratio of the right lung did not differ between groups at 2.33 t 0.25, 2.29 t 0.39, and 2.07 t 0.35 for the MAb 60.3-treated, saline-treated, and control groups, respectively. In addition there were no statistical differences between EVLW-to-plasma-to-BW 1251-albumin ratios between any of the groups. The median lavage-to-plasma 1251-albumin ratio was 0.0035 with a range of 0.053 for the
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INHIBITION
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NEUTROPHIL
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a range of 0.016 in the MAb 60.3-treated animals and to 0.0041 with a range of 0.0057 in the saline-treated animals. Arterial oxygerzation (sepsis). Arterial PO, decreased in both the saline- and MAb 60.3-treated septic groups. The decrease with time was significant by ANOVA; however, there was no difference between the two groups. In the saline-treated group, PO, decreased from 89.9 t 7.6 to 68 + 10.7 Torr, and in the MAb 60.3-treated group it decreased from 94.2 t 16.0 to 72.0 t 2.9 Torr at -24 h. Arterial PCO, and pH did not change significantly between the initial and final measurement. DISCUSSION
Saline
MAb 60.3
Normal
B
0.020 0.015
Q) E
-I i
0.002-
5
0 00
0
1
01
0
n 0.000
'
Saline
MAb 60.3
+
v
4
Normal
FIG. 6. Lung permeability index measured as ratio of 1251-labeled albumin in lavage fluid to 1251- albumi n in plasm a. Permeability indexes for animals given lipopolysaccharide (A) and for animals made septic (B) are shown. Lavage was performed 5 h after administration of lipopolysaccharide or 48 h after appendectomy in MAb 60.3- and salinetreated animals. Normal group received no treatment.
MAb 60.3 group, 0.002 with a range of 0.023 for the saline group, and 0.0007 with a range of 0.001 for the control group. The lavage-to-plasma 1251-albuminratio for all of these animals is shown in Fig. 6A. The three groups were significantly different when compared by the KruskalWallace H test, and the control group was different from both the MAb 60.3- and the saline-treated groups. There was no difference between the two groups given LPS. Permeability index (sepS;s).The ratio of 1251-albuminin bronchoalveolar lavage fluid to that in plasma for the two groups in this study together with normal controls is shown in Fig. 6B. This permeability index was significantly increased above normal in both the saline- and MAb 60.3-treated animals compared with the normal controls. The medial value for normal animals was 0.0007 with a range of 0.001 and increased to 0.0014 with
The CD18 MAb 60.3 allowed us to investigate the role of one pathway of neutrophil adherence and/or aggregation after LPS infusion in rabbits. There was a reduction in the resuscitative fluid requirement after LPS infusion in animals given MAb 60.3. The septic rabbits in the saline-treated group also required more fluid than the MAb 60.3-treated on the 1st day of sepsis. Because all septic animals had free accessto food and water overnight, it is not realistic to compare these animals after the 1st day. Survival was significantly increased at day 10 in the septic animals treated with MAb 60.3 compared with the saline-treated group. These results are consistent with neutrophils causing significant adverse effects as a result of sepsis or after endotoxin infusion and with the injury being CDlUCDl8 dependent. MAb 60.3 provided no protection to the lungs in these experiments as permeability of the lungs was not different between saline- and MAb 60.3-treated animals (either septic animals or after LPS infusion). Thus the protection provided by MAb 60.3 relative to decreased fluid requirements and increased survival suggestsprotection of some systemic organs. Cardiac output could not be maintained after LPS infusion in the saline-treated animals even with increased fluid administration. These results suggest that CDllI CDl8-dependent neutrophil injury may occur to the myocardium during endotoxic shock. Although other factors could explain these results (e.g., decreased preload), evidence of myocardial injury has been reported. Transvenous endomyocardial biopsies revealed intravascular stasis of neutrophils, focal neutrophil infiltration, and occasional myocyte necrosis by light microscopy in dogs given intraperitoneal Pseudomonas aeruginosas. Endothelial swelling and interstitial edema were seen by electron microscopy (22). The metabolic acidosis after infusion of LPS can be assumed to result from increased lactic acid production. Neither serum lactate nor electrolyte concentration (for ion-gap calculations) was measured in these experiments. This presumed lactic acidosis may have resulted from excess tissue production as a result of hypoperfusion or inadequate oxygen extraction or from diminished hepatic clearance of lactate. Determination of the cause of acidosis was beyond the scope of this study. From our results it would appear that neutrophil adherence and/or aggregation through a CDll/CDl8 pathway was not in-
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INHIBITION
OF
CD18DEPENDENT
volved in establishing the metabolic acidosis associated with endotoxic shock. Neutropenia after infusion of LPS and other stimuli is a common finding. In two separate studies the number of circulating radiolabeled neutrophils declined after intravenous LPS administration, and the neutrophils were shown to be sequestered in the pulmonary vascular bed (3,lZ). The experiments described here showed that the leukopenia seen after LPS infusion was not CDll/CD18 dependent because there was no difference between leukocyte counts in the saline-treated and MAb 60.3-treated rabbits. These findings are consistent with those seen by Lundberg and Wright (17) and Doerschuk et al. (6) after infusion of zymosan-activated serum or chemotactic peptide. Decreases in PMN deformability after activation with zy mosan-ac tivated plasma was demonstrated in vitro and proposed as a mechanism of neutropenia in vivo (15). This could account for the leukopenia we observed. These experi ments suggest that the endotheli al damage induced by perito nitis or LPS infusion was ameliorated by the CD18 MAb 60.3 in the systemic but not the pulmonary circulation. That is, permeability index increased in both septic animals and animals given LPS without regard to treatment with MAb 60.3. In addition, PO, decreased in the septic animals and was not different between saline and MAb 60.3 treatment. This suggests that adherence in the lung may result from a pathway that is CD18independent. Such a pathway has been characterized by Doerschuk et al. (7) in the lung and by Mileski et al. (19) in a macrophage-enriched peritoneum. Doerschuk et al. showed that neutrophil emigration in response to Streptococcus pneumoniae bacteria and hydrochloric acid instillation into the lung was CD18 independent. In the same study emigration toward the same stimuli in the systemic circulation was CD18 dependent. Mileski et al. showed that the mechanism of emigration into the peritoneum was dependent on whether macrophages were present. Macrophage enrichment converted the emigration mechanism from CD18 dependent to partially CD‘18 independent. Sepsis is the greatest single risk factor for development of ARDS in critically ill patients, and PMNs have been implicated as causing this syndrome. It is thought that the pulmonary edema seen in ARDS is the result of increased permeability in the lungs. In the present study sepsis caused an increase in the ratio of 12”1-albumin in bronchoalveolar lavage to 1251-albumin in plasma. This is consistent with an increase in permeability. Also, arterial PO, decreased with time in the septic animals, a finding consistent with respiratory distress. There was no difference in permeability index or arterial oxygenation in the animals treated with saline or MAb 60.3 after infusion of LPS. In a previous study from our laboratory we examined safety aspects of MAb 60.3 in abdominal sepsis by pretreating rabbits with MAb 60.3 at the time of devascularization and again at appendectomy (20). In that study we found equivalent mortality at 10 days and equivalent infectious complications (defined as abdominal abscess formation and/or wound infections) in saline- and MAb 60.3-treated animals. The present study was designed to
NEUTROPHIL
ADHERENCE
IN
1515
SEPSIS
examine the effectiveness of MAb 60.3 treatment of established abdominal sepsis. Both groups of animals in the present study were allowed to develop sepsis over 19 h and then divided into two groups for treatment with either saline or MAb 60.3. During development of sepsis, PMNs and monocytes were able to respond normally to the septic challenge by emigrating to the peritoneum in their host defense role. However, the cycle of inflammation was broken at 19 h with MAb 60.3 with an apparent reduction in the vascular injury. In summary, we examined the role of neutrophil adherence and aggregation in sepsis and after LPS infusion using CD18 MAb 60.3. Our findings suggest that neutrophils cause a physiological derangement during sepsis and endotoxemia and that MAb 60.3 therapy may prevent the cardiovascular dysfunction associated with these conditions. Also, CD18 MAb 60.3 improved survival in sepsis when given over an extended period. The authors thank W. Purcell and R. Knake-Othberg for superb technical assistance. The MAb 60.3 was a gracious gift from Dr. Patrick Beatty, Fred Hutchinson Cancer Center, Seattle, WA. This study was supported by National Heart, Lung, and Blood Institute Grants HL-43141 and HL-30542 and by a grant from the American Heart Association. Address for reprint requests: R. K. Winn, Dept. of Surgery, Harborview Medical Center (ZA-16), 325 Ninth Ave., Seattle, WA 98104. Received
9 December
1991; accepted
in final
form
1 May
1992.
REFERENCES 1. BEATTY, P. G., J. A. LEDBETTER, P. J. MARTIN, T. H. PRICE, AND J. A. HANSEN. Definition of a common leukocyte cell-surface antigen (Lp95 150) associated with diverse cell-mediated immune functions. J. Immunol. 131: 2913-2918, 1983. 2. BRUNING, J. L., AND B. L. KINTZ. ComputationaL Handbook of Statistics. Glenview, IL: Scott, Foresman, 1977. 3. CYBULSKY, M. I., AND H. Z. MOVAT. Experimental bacterial pneumonia in rabbits: polymorphonuclear leukocyte margination and sequestration in rabbit lungs and quantitation and kinetics of ‘%rlabeled polymorphonuclear leukocytes in E. coli-induced lung lesions. Exp. Lung Res. 4: 47-66, 1982. 4. DESCH, C., N. KOVACH, W. PRESENT, C. BROYLES, AND J. HARLAN. Production of human tumor necrosis factor from whole blood ex vivo. Lymphokine Res. 8: 141-146, 1989. 5. DIENER, A. M., P. G. BEATTY, H. D. OCHS, AND J. M. HARLAN. The role of neutrophil membrane glycoprotein 150 (GP-150) in neutroPhil-mediated endothelial cell injury in vitro. J. Immunol. 135: 537543, 1985. 6. DOERSCHUK, C. M., D. ENGLISH, J. M. HARLAN, AND J. C. HOGG. The role of CD18 in neutropenia and neutrophil (PMN) sequestration induced by infusion of activated plasma (Abstract). FASEB J. 4: 496A, 1990. 7. DOERSCHUK, C. M., R. K. WINN, H. 0. COXSON, AND J. M. HARLAN. CD18-dependent and -independent mechanisms of neutrophi1 emigration in the pulmonary and systemic microcirculation of rabbits. J. Immunol. 144: 2327-2333, 1990. 8. FAIST, E., A. E. BAUE, H. DITTMER, AND G. HEBERER. Multiple organ failure in polytrauma patients. J. Trauma 23: 775-787, 1983. 9. FRY, D. E., L. PEARLSTEIN, R. L. FULTON, AND H. C. POLK. Multiple system organ failure: the role of uncontrolled infection. Arch. Surg. 115: 136-140, 1980. 10. GORIS, R. J. A., T. P. TE BOEKHORST, J. K. S. NUYTINCK, AND J. S. F. GIMBRERE. Multiple organ failure: generalized autodestructive inflammation? Arch. Surg. 120: 1109-1115, 1985. 11. HARLAN, J. M., B. R. SCHWARTZ, W. J. WALLIS, AND T. H. POHLMAN. The role of neutrophil membrane proteins in neutrophil emigration. In: Leukocyte Emigration and Its Sequelae, edited by H. Z. Movat. Krager: Basel, 1987, p. 94-104. 12. HASLETT, C., G. S. WORTHEN, P. C. GICLAS, D. C. MORRISON, J. E. HENSON, AND P. M. HENSON. The pulmonary vascular sequestra-
Downloaded from www.physiology.org/journal/jappl at Univ of Cincinnati MSB R005B Box 574 (129.137.005.042) on February 12, 2019.
1516
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
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OF
CDl8DEPENDENT
tion of neutrophils in endotoxemia is initiated by an effect of endotoxin on the neutrophil in the rabbit. Am. Reu. Respir. Dis. 136: 9-181987. HEFLIN, A. C., AND K. L. BRIGHAM. Prevention by granulocyte depletion of increased vascular permeability of sheep lung following endotoxemia. J. Clin. Invest. 68: 1253-1260, 1981. HYERS, T. M., AND A. A. FOWLER. Adult respiratory distress syndrome: causes, morbidity, and mortality. Federation Proc. 45: 2529, 1986. INANO, H., AND C. M. DOERSCHUK. The effect of zymosan-activated plasma on neutrophil (PMN) deformability in vitro (Abstract). Am. Rev. Respir. Dis. 143: 329A, 1991. LAUFE, M. D., R. H. SIMON, A. FLINT, AND J. F. KELLER. Adult respiratory distress syndrome in neutropenic patients. Am. J. Med. 80:1022-1026,1986. LUNDBERG, C., AND S. D. WRIGHT. Relation of the CDll/CD18 family of leukocyte antigens to the transient neutropenia caused by chemoattractants. Blood 76: 1240-1245, 1990. MAUNDER, R. J., R. C. HACKMAN, E. RIFF, R. K. ALBERT, AND S. S. SPRINGMEYER. Occurrence of the adult respiratory distress syndrome in neutropenic patients. Am. Rev. Respir. Dis. 133: 313-316, 1986. MILESKI, W., J. HARLAN, C. RICE, AND R. WINN. Streptococcus pneumoniae-stimulated macrophages induce neutrophils to emigrate by a CD18-independent mechanism of adherence. Circ. Shock 31: 259-267,199O. MILESKI, W. J., R. K. WINN, J. M. HARLAN, AND C. L. RICE. Transient inhibition of neutrophil adherence with the anti-CD18 monoclonal antibody 60.3 does not increase mortality rates in abdominal sepsis. Surgery 109: 497-501, 1991. MILESKI, W. J., R. K. WINN, N. V. VEDDER, T. H. POHLMAN, J. M. HARLAN, AND C. L. RICE. Inhibition of CD18-dependent neutrophil adherence reduces organ injury after hemorrhagic shock in primates. Surgery 108: 205-212, 1990. NATANSON, C., R. E. CUNNION, D. A. BARRETT, K. W. PEART, R. L. DANNER, J. J. CONKLIN, T. J. MACVITTIE, R. I. WALKER, V. J. FERRANS, AND J. E. PARRILLO. Reversible myocardial dysfunction in a canine model of septic shock is associated with myocardial microcirculatory damage and focal neutrophil infiltration (Abstract). Clin. Res. 34: A639, 1986. OGNIBENE, F. P., S. E. MARTIN, M. M. PARKER, T. SCHLESINGER,
NEUTROPHIL
24. 25.
26.
27.
28.
29. 30.
31.
32.
33.
34.
ADHERENCE
IN
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P. ROACH, C. BURCH, J. H. SELHAMER, AND J. E. PARRILLO. Adult respiratory distress syndrome in patients with severe neutropenia. N. Engl. J. Med. 315: 547-551, 1986. PEARCE, M. L., J. YAMASHITA, AND J. BEAZELL. Measurement of pulmonary edema. Circ. Res. 41: 482-488, 1965. PINGLETON, W. W., J. J. COALSON, AND C. A. GUENTER. Significance of leukocytes in endotoxic shock. Exp. Mol. Puthol. 22: 183194, 1975. REMINGTON, R. D. AND M. A. SCHORK. Statistics With Applications to the Biological and Health Sciences. Englewood Cliffs, NJ: Prentice Hall, 1970. SACKS, T., C. F. MOLDOW, P. R. CRADDOCK, T. K. BOWERS, AND H. S. JACOB. Oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes. J. Clin. Invest. 61: 11611167, 1978. SHASBY, D. M., S. S. SHASBY, AND M. J. PEACH. Granulocytes and phorbol myristate acetate increase permeability to albumin of cultured endothelial monolayers and isolated perfused lungs. Role of oxygen radicals and granulocytes. Am. Rev. Respir. Dis. 127: 72-76, 1983. SPRINGTHALL, R. C. Basic Statistical Analysis. Reading, MA: Addison-Wesley, 1982. VEDDER, N. B., B. W. FOUTY, R. K. WINN, J. M. HARLAN, AND C. L. RICE. Role of neutrophils in generalized reperfusion injury associated with resuscitation from shock. Surgery 106: 509-516, 1989. VEDDER, N. B., R. K. WINN, C. L. RICE, E. CHI, K.-E. ARFORS, AND J. M. HARLAN. A monoclonal antibody to the adherence promoting leukocyte glycoprotein CD18 reduces organ injury and improves survival from hemorrhagic shock and resuscitation in rabbits. J. Clin. Invest. 81: 939-944, 1988. WELSH, C. H., D. C. LIEN, G. S. WORTHEN, P. M. HENSON, AND J. V. WEIL. Endotoxin-pretreated neutrophils increase vascular permeability in dogs. J. Appl. Physiol. 66: 112-119, 1989. WELSH, C. H., D. C. LIEN, G. S. WORTHEN, AND J. V. WEIL. Pentoxifylline decreases endotoxin-induced pulmonary neutrophil sequestration and extravascular protein accumulation in the dog. Am. Rev. Respir. Dis. 138: 1106-1114, 1988. WINN, R., R. MAUNDER, E. CHI, AND J. HARLAN. Neutrophil depletion does not prevent lung edema after endotoxin infusion in goats. J. Appl. Physiol. 62: 116-121, 1987.
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