Serum Macrophage Inflammatory Protein-2 Research Articles

Protective effect of quercetin on acute lung injury in rats with sepsis and its influence on ICAM-1 and MIP-2 expression.

This study aimed to explore the protective effect of quercetin on acute lung injury (ALI) in rats with sepsis and the related mechanism. Rats were administered different doses of quercetin intraperitoneally, and blood samples and lung tissue were collected at 24 h after treatment. Arterial blood gases, lung water content, protein content, and cell counts in bronchoalveolar lavage fluid (BALF) were measured. Morphological changes in lung tissue pathology were observed under a light microscope. Serum intercellular adhesion molecule (ICAM)-1 and macrophage inflammatory protein 2 (MIP-2) levels were detected and ICAM-1 and MIP-2 mRNA expression in lung tissue was determined. Compared with that in the control model group, arterial blood gases, lung water content, protein content, and cell counts in BALF improved in the high- and low-dose quercetin groups (P < 0.05), with maximal improvement observed for the high-dose quercetin (P < 0.05). Lesions on the lungs improved in the high- and low-dose quercetin groups than those in the control model group, and the high-dose quercetin group showed better improvement than the low-dose group (P < 0.05). Compared with that in the sham-operated group, both serum and lung tissue ICAM-1 and MIP-2 expression increased significantly in the model group (P < 0.05). The quercetin groups presented lower ICAM-1 and MIP-2 expression than the control model group, with the lowest expression observed in the high-dose group (P < 0.05). Quercetin may protect against ALI in rats with sepsis by inhibiting ICAM-1 and MIP-2 expression.

A Murine Model Of Hemorrhagic Shock/Resuscitation Demonstrates Transfusion Related Acute Lung Injury (TRALI) Induced By Stored Platelet Transfusions

Transfusion related acute lung injury (TRALI) is one of the leading causes of transfusion fatalities. There have been several animal models of TRALI developed including ex vivo lung models demonstrating the importance of human anti-neutrophil antibodies in TRALI and in vivo transfusion models showing how biological response modifiers can induce recipient lung damage. In addition, an in vivo antibody-mediated TRALI model has also been established and has shown close similarities with human TRALI. Most of the animal models have a two-hit paradigm where the first hit is delivered in the form of a noxious substance such as lipopolysaccharide and the second hit is a transfusion of either a blood product or an antibody. Here we describe a novel clinically relevant two-hit model where the first hit is hemorrhagic shock followed by a second hit in the form of a platelet transfusion. Severe combined immunodeficient (SCID) mice were anesthetized and had half of their total blood volume was removed via a carotid cannula to instigate hypovolemic shock. After one hour of shock, the mice were reperfused with either Ringer's lactate solution or fresh or three day aged platelets. 34-1-2s, a monoclonal anti-mouse MHC class I antibody known to induce TRALI was used as a positive reperfusion control. Two hours following reperfusion, the mice were sacrificed and blood was obtained via cardiac puncture in order to measure serum levels of the neutrophil chemoattractant macrophage inflammatory protein-2 (MIP-2). Lungs were removed to determine the percentage of accumulated neutrophils and to measure edema. Compared with control mice reperfused with Ringer's lactate solution or mice administered fresh platelets, serum MIP-2 levels in mice administered with three day aged platelets were significantly elevated and were comparable to those observed in mice infused with 34-1-2s. Pulmonary neutrophil accumulation in the mice transfused with 3 day stored platelets was also elevated to levels observed in 34-1-2s treated mice. Pulmonary edema as measured by lung Wet/Dry ratios was elevated in mice receiving the 3 day aged platelets. In vivo administration of a MIP-2 antagonist (SB225002, 4mg/kg) intraperitoneally immediately before reperfusion significantly reduced all TRALI symptoms. These results show that an animal model of hypovolemic shock exhibits TRALI upon reperfusion with stored platelets and is dependent on MIP-2 production. This clinically-relevant model will be valuable in studying potential therapeutic interventions to prevent TRALI following transfusions, Disclosures:No relevant conflicts of interest to declare.

Role of MexA-MexB-OprM efflux pump system in chronic Pseudomonas Aeruginosa pulmonary infection in mice

In order to investigate the role of the MexA-MexB-OprM efflux pump system in the pathogenesis of Pseudomonas aeruginosa (PA)-induced pulmonary infection, pulmonary infection models were established by intratracheal injection of K767 (wild type), nalB (MexA-MexB-OprM up-regulated mutant), and ΔmexB (knockout) strains, separately. All mice were treated with Meropenem (intraper Δ itoneal injection, 100 mg/kg body weight, twice every day), and strain-related pathology, bacteria count, cytokine level, myeloperoxidase (MPO, indicator of neutrophil recruitment) activity, and macrophage inflammatory protein-2 (MIP-2) expression were evaluated at early (3rd day post-infection) and late (7th and 14th day post-infection) stages of infection. E-test showed that ΔmexB was more significantly Δ sensitive to panipenan (ETP), meropenem (MP) and imipenem (IP) than K767 and nalB strains. There was no significant difference in sensitivity to cefepime (TM) among the three stains. In contrast to the K767 and nalB groups, the ΔmexB group showed decreased bacteria burden over time and less exte Δ nsive pathological change. Additionally, MPO activity and levels of inflammatory cytokines (IL-1b, IL-12, and TNF-α) were increased at the early stage (day 3) and decreased at the later stage (day 14). Serum MIP-2 expression level was steadily increased in all three groups from early to late stages, but significantly higher in ΔmexB group than in K767 and nalB groups ( Δ P<0.05). In conclusion, the MexA-MexB-OprM efflux pump system might play an important role in PA-induced chronic pulmonary infection. High expression of the MexA-MexB-OprM efflux pump could increase antibacterial resistance and promote infection.

A Serotype 3 Pneumococcal Capsular Polysaccharide-Specific Monoclonal Antibody Requires Fcγ Receptor III and Macrophages To Mediate Protection against Pneumococcal Pneumonia in Mice

Antibodies to pneumococcal capsular polysaccharide (PPS) are required for PPS-based vaccine-mediated protection against Streptococcus pneumoniae. Previous work established that 1E2, a mouse IgG1 to PPS3 that does not induce serotype 3 (ST3) S. pneumoniae killing by phagocytes in vitro, protects mice from death after intranasal infection with ST3, but its efficacy was abrogated in FcγR (F common gamma receptor)-deficient mice. In this study, we determined whether 1E2 efficacy against pulmonary ST3 infection requires FcγRIII. 1E2 did not protect FcγRIII-deficient (FcγRIII(-/-)) mice. Studies of the mechanism of 1E2-mediated effects showed that it resulted in a marked reduction in lung inflammation in ST3-infected wild-type (Wt [C57BL/6]) mice that was abrogated in FcγRIII(-/-) mice. 1E2 had no effect on early bacterial clearance in the lungs of ST3-infected Wt, FcγRIIB(-/-), or FcγRIII(-/-) mice, but it reduced levels of bacteremia and serum macrophage inflammatory protein-2) (MIP-2), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) in Wt and FcγRIIB(-/-) mice, strains in which it is protective. As previous work showed that neutrophils were dispensable for 1E2 efficacy, we investigated whether macrophages are required for 1E2 efficacy against intranasal infection with ST3 and found that its efficacy was abrogated in Wt mice depleted of macrophages intranasally. In vitro studies revealed that1E2 promoted ST3 internalization by naïve alveolar macrophages but did not induce early intracellular killing. Macrophages from 1E2-treated ST3-infected mice studied ex vivo exhibited more apoptosis than those from FcγRIII(-/-) mice. These findings suggest that 1E2 mediates protection against ST3 in mice by affecting the inflammatory response, perhaps in part via macrophage apoptosis, rather than by inducing early bacterial clearance.

CD40L is not involved in acute experimental pancreatitis

Recent data suggest that platelets not only control thrombosis and hemostasis but may also regulate inflammatory processes such as acute pancreatitis. However, the specific role of platelet-derived mediators in the pathophysiology of acute pancreatitis is not known. Herein, we examined the role of CD40 ligand (CD40L, CD154) in different models of acute pancreatitis. Acute pancreatitis was induced by repetitive caerulein administration (50 μg/kg, i.p.) or infusion of sodium taurocholate (5%–10 μl) into the pancreatic duct in wild-type C57BL/6 and CD40L-deficient mice. Neutrophil infiltration, myeloperoxidase (MPO), macrophage inflammatory protein-2 (MIP-2) levels, acinar cell necrosis, edema and hemorrhage in the pancreas as well as serum amylase activity and lung levels of MPO were quantified 24 h after induction of acute pancreatitis. Caerulein and taurocholate challenge caused a clear-cut pancreatic damage characterized by increased acinar cell necrosis, neutrophil infiltration, focal hemorrhage, edema formation as well as increased levels of serum amylase and MIP-2 in the pancreas and lung MPO and histological damage. Notably, CD40L gene-deficient animals exhibited a similar phenotype as wild-type mice after challenge with caerulein and taurocholate. Similarly, administration of an antibody directed against CD40L had no effect against acute pancreatitis. Our data suggest that CD40L does not play a functional role in experimental acute pancreatitis. Thus, other candidates than CD40L needs to be explored in order to identify platelet-derived mediators in the pathophysiology of acute pancreatitis.

Procarboxypeptidase R Deficiency Causes Increased Lethality in Concanavalin A-Induced Hepatitis in Female Mice

Carboxypeptidase R (CPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), is an enzyme generated by proteolytic cleavage of its zymogen (proCPR). CPR removes the C-terminal arginine from inflammatory peptides such as C3a and C5a, bradykinin, enkephalin, and the thrombin-cleaved N-terminal fragment osteopontin (cleaved N-OPN). In the mouse model of concanavalin A (Con A)-induced immune-mediated fulminating hepatitis, cleaved N-OPN is one of the important peptides that induce the production of chemokines or cytokines. In the current study using proCPR deficient mice, we showed that injection of Con A into the mouse tail vein can induce a significantly higher lethality in proCPR-deficient female but not in male mice. Furthermore, a lack of CPR activity increased serum macrophage inflammatory protein-2 (MIP-2) and high-mobility group box 1 (HMGB1) levels after Con A injection. These in vivo findings suggest that CPR helps to protect against Con A-induced hepatitis.

Suppressive effect of phosphodiesterase type 4 inhibition on systemic inflammatory responses after cardiopulmonary bypass

Cardiopulmonary bypass (CPB) induces excessive production of endogenous proinflammatory mediators such as cytokines and elastase, which are responsible for the subsequent development of systemic inflammatory response syndrome (SIRS). In this study, we investigated the protective effect of rolipram against SIRS after CPB. Rats were divided into three groups (n = 5 in each): control (C), rolipram (R), and sham (S). Rats in groups C and R underwent CPB for 60 min followed by 60 min of observation, while those in group S were observed for 120 min without CPB. In group R, 40 microg/kg/min of rolipram was intravenously administered throughout the experiment. CD11b expression on neutrophils was analyzed using flow cytometry. Serum concentrations of tissue necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), macrophage inflammatory protein 2 (MIP-2), and elastase were also determined. CD11b expression at the end of the experiment was unchanged from the initial value in group R, whereas that in group C increased to almost double, and that in group S also showed a slight increase (P < 0.01). Serum TNF-alpha levels in groups R and S were lower than those observed in group C (P < 0.05). Serum IL-1beta and MIP-2 levels in groups C and R tended to be higher than those in group S, although the difference was not statistically significant. Regarding elastase, group R showed a significantly lower value than group C and a higher value than group S (P < 0.05). Phosphodiesterase type 4 inhibition seems to suppress CPB-induced SIRS through the regulation of proinflammatory mediators in this rat model.

Coagulation-Dependent Gene Expression and Liver Injury in Rats Given Lipopolysaccharide with Ranitidine but Not with Famotidine

In an animal model of drug idiosyncrasy, rats cotreated with nonhepatotoxic doses of lipopolysaccharide (LPS) and ranitidine (RAN) develop hepatocellular injury, whereas rats treated with LPS and famotidine (FAM) do not. The coagulation system and neutrophils (PMNs) are requisite mediators of LPS/RAN-induced liver injury. We tested the hypothesis that unique gene expression in LPS/RAN-treated rats requires coagulation system activation and that these changes are absent in rats given LPS and FAM. Rats were treated with a nonhepatotoxic dose of LPS (44.4 x 10(6) endotoxin units/kg i.v.) or its vehicle, and then 1 h later, they were treated with heparin (3000 U/kg) or its vehicle. One hour thereafter, they were given RAN (30 mg/kg), FAM (6 mg/kg, a pharmacologically equiefficacious dose, or 28.8 mg/kg, an equimolar dose), or vehicle (i.v.). They were killed 2 or 6 h after drug treatment for evaluation of hepatotoxicity, coagulation system activation, and liver gene expression (2 h only). Statistical filtering of gene array results and real-time polymerase chain reaction identified groups of genes expressed in LPS/RAN-treated rats but not LPS/FAM-treated rats that were either changed or unchanged by heparin administration. For example, LPS/RAN-induced mRNA expression of the inflammatory mediators interleukin-6, cyclooxygenase-2, and macrophage inflammatory protein-2 (MIP-2) was reduced by anticoagulation. Enhancement of serum MIP-2 and plasminogen activator inhibitor-1 concentrations in LPS/RAN-treated rats was prevented by anticoagulation. The results suggest cross-talk between hemostasis-induced gene expression and inflammation (e.g., PMN function) in the genesis of hepatocellular injury in LPS/RAN-treated rats. In contrast, neither the expression of such genes nor hepatocellular necrosis occurred in rats treated with LPS/FAM.

Neutralization of macrophage inflammatory protein-2 blocks the febrile response induced by lipopolysaccharide in rats

1. This study was aimed to test the hypothesis that macrophage inflammatory protein-2 (MIP-2), a powerful chemotactic cytokine for neutrophils, plays a role in bacterial endotoxin fever. 2. The effect of specific anti-rat MIP-2 antibodies on lipopolysaccharide (LPS)-induced fever was tested. Intraperitoneal injection of LPS resulted in a biphasic fever and a significant increase in serum MIP-2 and prostaglandin (PG) E 2 levels which correlated with the start of fever. Intraperitoneal anti-MIP-2 (500 μg/kg) did not affect the body core temperature of unrestrained rats, but markedly attenuated LPS-induced fever. 3. Treatment with the cyclooxygenase inhibitor ibuprofen (10 mg/kg) resulted in a significant attenuation of LPS-induced fever and a significant decrease of MIP-2 and PGE 2 production. 4. These results indicate that LPS fever in rats is, at least, in part dependent on mechanisms involving neutrophils chemotaxis, and that MIP-2 may be an important mediator in the genesis of fever via prostaglandin-dependent pathways.

Effects of immunoglobulin upon murine myocarditis caused by influenza A virus: superiority of intact type to F(ab')2 type.

Influenza viruses play the largest role in the worldwide epidemiology of infectious diseases. Management of some inflammatory disease (eg, Kawasaki disease) with immunoglobulin has been demonstrated to be effective. We examined the effects of intact type and F(ab')2 type of immunoglobulin preparations upon murine influenza A virus myocarditis in mice. In vitro study showed that intact type and F(ab')2 type of immunoglobulin preparations exhibit antiviral activities against many substrains of influenza virus and other cardiotropic viruses. Dose-dependent suppression of an influenza A virus (NWS) was demonstrated by management with both intact immunoglobulin and F(ab')2 fragments of immunoglobulin. The dose inhibiting 50% of plaques was the same between intact type and F(ab')2 type (both 0.0002 mg/dl). Intact immunoglobulin, but not F(ab')2 fragments of immunoglobulin, suppressed serum macrophage inflammatory protein-2 (MIP-2) productions in influenza A virus-infected macrophages in vitro, which is a murine counterpart of interleukin-8. This suppression of MIP-2 production by intact immunoglobulin treatment was blocked by a specific Fc receptor (Fc gamma III/II receptor) antibody pretreatment. Intact immunoglobulin or F(ab')2 fragments of immunoglobulin were administered to virus-inoculated A/J mice intraperitoneally daily, starting simultaneously with virus inoculation (Experiment I) and 2 days after the virus inoculation (Experiment II), until 10 days after virus inoculation. In Experiment I, survival was higher in treated than in control mice; intact type and F(ab')2 type immunoglobulins administration completely suppressed the development of myocarditis. In Experiment II, survival rate was significantly higher and myocarditis was less severe in intact immunoglobulin-treated mice, but not in F(ab')2 fragments-treated mice compared with untreated mice. Serum neutralizing antibody titers in treated mice were significantly higher compared with untreated mice in Experiments I and II. In addition, serum MIP-2 concentrations in intact immunoglobulin-treated mice, but not in F(ab')2 fragments-treated mice, were lower compared with untreated mice in Experiment II. Immunoglobulin therapy suppresses influenza A virus myocarditis by increasing neutralizing antibody titers and the suppression of myocardial virus activities. From the standpoint of suppression of MIP-2 concentrations, intact type is superior to F(ab')2 type. Thus, immunoglobulin treatment may be promising for prevention of influenza virus myocarditis.

Up-regulation of macrophage inflammatory protein-2 and complement 3A receptor by the trichothecenes deoxynivalenol and satratoxin G

The trichothecenes are a group of mycotoxins that target leukocytes and have a wide range of immunomodulatory effects. Differential display analysis was applied to assess the effects of the trichothecenes deoxynivalenol (vomitoxin, DON) and satratoxin G (SG), on mRNA in the RAW 264.7 macrophage cell line. Cells were incubated with DON (1 μg/ml) or SG (5 ng/ml) for 2 h and total RNA then subjected to RT-PCR with a set of oligo(dT) primers. Resultant cDNA was amplified using an oligo (dT) downstream primer and an arbitrary decanucleotide upstream primer to make 35S-labeled PCR products. After separation of the products in denaturing polyacrylamide gel, 23 differentially expressed cDNA fragments were isolated and sequenced. Two of these were identified as known genes, namely, macrophage inflammatory protein-2 (MIP-2), a potent neutrophil chemoattractant involved in tissue injury and inflammation, and complement 3a receptor (C3aR), a proinflammatory mediator. Both MIP-2 and C3aR mRNAs were up-regulated by DON while only MIP-2 mRNA was induced by SG. Using commercially available antibodies, MIP-2 protein was also found to be induced by both DON and SG in RAW 264.7 cell cultures. When mice were treated with DON (12.5 mg/kg), splenic MIP-2 mRNA and serum MIP-2 levels were increased. MIP-2 mRNA and serum MIP-2 levels were synergistically increased when mice were co-treated with DON and LPS. Up-regulation of MIP-2 and C3aR are consistent with previous reports of trichothecene-induced inflammatory gene up-regulation and suggest that the specific genes affected may depend on trichothecene structures.

Ischemic preconditioning decreases C-X-C chemokine expression and neutrophil accumulation early after liver transplantation in rats.

Polymorphonuclear neutrophil (PMN) plays a major role in liver ischemia/reperfusion injury. Protective effect of ischemic preconditioning (IP) has been confirmed in liver ischemia/reperfusion injury. The purpose of this study was to investigate the effect of IP on C-X-C chemokine expression and PMNs recruitment early after liver transplantation. Male Sprague-Dawley rats were used as donors and recipients of orthotopic liver transplantation (OLT). The donor liver was stored 24 hours in University of Wisconsin (UW) solution at 4 degrees pre-implantation. IP was done by clamp of the portal vein and hepatic artery of the donor liver for 10 minutes followed by reperfusion for 10 minutes before harvesting. The neutrophilic infiltration in liver was quantified using a myeloperoxidase (MPO) assay. Intragraft expression of macrophage inflammatory protein-2 (MIP-2) mRNA was investigated with in situ hybridization. The serum levels of MIP-2 and tumor necrosis factor (TNF)-alpha were also monitored. After liver transplantation without IP, the hepatic MPO increased significantly compared with sham operated group. In IP group, PMN in liver indicated by MPO was reduced significantly. In situ hybridization showed no MIP-2 mRNA in sham group but dramatic expression in hepatocytes in non-IP group. In IP group, MIP-2 mRNA was significantly down-regulated. Similarly, serum MIP-2 and TNF-alpha levels were significantly elevated in non-IP group and both were reduced in IP group. IP might protect graft liver from preservation-reperfusion injury after OLT through down-regulating C-X-C chemokine expression of hepatocytes, and alleviating PMNs recruitment after reperfusion.

Eicosapentaenoic acid supplementation reduces tumor volume and attenuates cachexia in a rat model of progressive non-metastasizing malignancy.

Eicosapentaenoic acid (EPA) has been shown to have anti-inflammatory and tumor growth inhibitory effects clinically and experimentally; evidence also supports the role of EPA in attenuating cancer-associated weight loss, but the mechanisms of these effects remain to be defined. As the liver plays a central role in modulating nutritional status and the cachexia syndrome, we examined the liver and nutritional parameters indicative of cachexia along with the tumor volume in response to oral EPA supplementation in a rat model of progressive non-metastasizing malignancy. Fischer 344 rats implanted with the methylcholanthrene-induced fibrosarcoma (MCA) were trained to meal-feed with access to food from 8:00 PM to 8:00 AM and water ad libitum. On day 13, rats were randomly divided into 3 study groups: (1) 5.0 g/kg per day EPA plus 10 IU vitamin E/g fat, (2) 5.0 g/kg per day corn oil plus 10 IU vitamin E/g fat, and (3) 5.0 g/kg per day saline plus 10 IU vitamin E/g saline. The treatment was delivered via oral gavage twice daily. The animals were killed on day 29, and serum plus tissues (ie, liver and lung) were collected and frozen for analysis. Parameters evaluated include the following: tumor volume, weight loss, liver weight, total liver protein, liver lipid content, serum albumin content, and macrophage inflammatory protein-2 (MIP-2) levels. EPA-treated rats showed a reduction in tumor volume compared with corn oil (25% reduction, p < .01) and saline (33% reduction, p < .01) animals. EPA rats also demonstrated increased liver weight (p < .01) and total liver protein levels (p < .03) over saline-treated animals. EPA- and corn oil-treated rats received more calories than the saline group because of the dietary fat treatments (p < .01) and had elevated lipid content in their livers (p = .05 and p = .04, respectively) compared with saline rats. Serum albumin (a marker of liver function) and MIP-2 levels (a marker of the hepatic acute phase response) were not different between treatment groups. EPA supplementation resulted in a dramatic reduction of tumor volume and mild improvements in weight maintenance. In addition, EPA-treated animals demonstrated increased total liver protein and serum protein levels. Regression analyses showed that the weight and protein differences between treatment groups were not correlated with individual tumor volumes. The increase in liver and serum protein was not explained by differences in albumin or MIP-2. We conclude that the tumor growth inhibitory effects and anticachexiogenic effects of EPA are independent phenomena, and the effects on cachexia may be related to increased levels of undefined protein(s) in the liver and serum. To our knowledge, this is the first study to demonstrate the effects of EPA in the MCA fibrosarcoma model and is also novel in its evaluation of EPA as an anticachexiogenic therapy in progressive non-metastasizing malignancy. Further studies may identify the protein(s) elevated in the liver and the mechanisms for the development of EPA nutritional therapies for the treatment of progressive malignancies.

Antibody-mediated Immune Enhancement in Coxsackievirus B3 Myocarditis

C. K ISHIMOTO, M. K UROKAWA and H. O CHIAI. Antibody-mediated Immune Enhancement in Coxsackievirus B3 Myocarditis. Journal of Molecular and Cellular Cardiology (2002) 34, 1227–1238. The aim of the present study was to explore the contribution of antibody-mediated immune enhancement in coxsackievirus B3(CB3) infection. Murine macrophage-like P388D1 cells were exposed to various concentrations of anti-CB3 immunoglobulin G (anti-CB3 IgG) or the Fab fragment of anti-CB3 IgG, and were infected with CB3 in Experiment I. High concentrations of anti-CB3 IgG showed a virus-neutralizing activity; however, a subneutralizing antibody concentration of IgG significantly enhanced virus replication. This infectious enhancement was blocked not only by the pretreatment of heat-aggregated γ-globulin but by a specific Fc receptor (Fc γ III/II receptor) antibody treatment. In contrast, the Fab fragment of anti-CB3 IgG did not enhance CB3 infection, but showed a rational neutralizing activity to CB3. These findings suggest the presence of Fc receptor mediated enhancement of CB3 infection in vitro. In Experiment II, C3H/He mice were inoculated with various amounts of an amyocarditic variant of CB3 followed 15 days later by myocarditic CB3. By this rechallenge, myocarditis was not induced in the mice with high neutralizing antibody titers. There was an inverse relationship between preexisting neutralizing antibody titers and the severity of myocarditis. The severity of myocarditis and myocardial CB3 titers, however, were markedly enhanced in the mice with a subneutralizing level of immunity compared to those with no immunity. The distribution of myocardial Fc receptor-bearing cells and serum macrophage inflammatory protein-2 levels paralleled the severity of myocarditis. By another virus rechallenge in Experiment III, enhanced infection of CB3 was not observed in vivo. These findings suggest that antibody-mediated immune enhancement might be involved in the pathogenesis of CB3 myocarditis.

Effect of cold-ischemia time on C-X-C chemokine expression and neutrophil accumulation in the graft liver after orthotopic liver transplantation in rats

The precise mechanisms leading to polymorphonuclear neutrophil (PMN) recruitment and activation in the extended cold-preserved liver after transplantation are not yet fully understood. We histologically evaluated the number of accumulated PMNs in graft livers, with varying time periods of cold ischemia (1, 6, and 24 hr in University of Wisconsin solution at 4 degrees C), after liver transplantation in rats. Intragraft expression of macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) mRNA, as well as immunohistochemical expression of MIP-2 and CINC in the graft liver, were investigated after reperfusion. The levels of MIP-2 and CINC in the hepatic vein, and tumor necrosis factor (TNF)-alpha, which stimulates these chemokine production, were also monitored. The number of accumulated PMNs in sinusoids significantly increased in the 24-hr cold-ischemia group within 3 hr after reperfusion, compared with the 1-hr and 6-hr groups. Serum MIP-2 levels in the 24-hr group significantly increased at 3, 6, and 12 hr after reperfusion, compared with the other groups. Intragraft MIP-2 mRNA was also up-regulated to a greater extent in the 24-hr group. Similarly, serum CINC levels in the 24-hr group significantly increased at 3 hr, compared with the 1-hr group. CINC mRNA also increased as cold-ischemia time was prolonged. Immunohistochemical staining revealed that hepatocytes were the main source of both MIP-2 and CINC protein. In addition, TNF-alpha in the hepatic vein was detected only in the 24-hr group after reperfusion. Extended cold preservation of the graft liver might up-regulate MIP-2 and CINC expression of hepatocytes, most probably through elevated TNF-alpha, and might contribute to PMN recruitment and activation after reperfusion.

Neutrophil Depletion and Chemokine Response after Liver Ischemia and Reperfusion

Neutrophils play a major role in the hepatic microvasculature following liver ischemia and reperfusion (I/R). Leukocyte cytokine chemoattractants (chemokines) are produced by neutrophils and cause neutrophil activation in I/R injury. We examined the role of neutrophils in the production of chemokines in the liver and lung inflammatory response following liver I/R. C57BL/6 mice were subjected to partial liver ischemia for 90 min. Four groups of animals were included: sham group, sham group with neutrophil depletion, ischemic control group, and ischemic control with neutrophil depletion. We evaluated at 3 h liver injury measurements, serum macrophage inflammatory protein-2 (MIP-2) and macrophage inflammatory protein-1 alpha (MIP-1alpha) chemokines, liver and lung tissue myeloperoxidase (MPO), and liver and lung histology. Statistical analysis included analysis of variance (ANOVA), and Student-Newman-Keuls and Kruskal-Wallis multiple comparison Z-value tests. Ischemic controls showed a significant increase in liver enzyme levels along with statistically significant higher liver and lung MPO activity values than the rest of the other groups (p < .05). MIP-2 values were higher in the ischemic control group when compared to the ischemic neutrophil depleted group. MIP-1alpha levels showed opposite results, being significantly lower (p < .05) in the ischemic control as compared to the neutrophil-depleted group. Improved liver and lung histopathological features were observed in the ischemic neutrophil depleted group when compared to the ischemic control group. Our study confirmed the key role of neutrophils in liver I/R injury and appeared to suggest some relationship between neutrophils and the production of certain chemokines, such as MIP-1alpha, which had an inverse relationship in the absence of neutrophils. Further studies will confirm the validity of these preliminary observations.

Lipo prostaglandin E1 reduces the production of CXC chemokines in endotoxin-induced rat liver injury

The present study attempted to assess the effect of prostaglandin E1 (PGE1) incorporated into lipid microspheres (Lipo PGE1) on chemokine production in endotoxin-induced rat liver injury. Male Wistar rats weighing 200–250 g were injected with 2 mg lipopolysaccharide (LPS) per kg intravenously. Lipo PGE1 was administered simultaneously at various concentrations (0.002, 0.02, 0.2, 2 μg/kg) in the tail vein. Blood samples and liver specimens were taken from the rats at 1, 3, 8, 12 and 24 h after injection with LPS alone or with LPS and Lipo PGE1. Serum macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) levels were measured by the enzyme-linked immunosorbant assay using the corresponding antibodies. Liver specimens were fixed, and the number of neutrophils that had infiltrated each liver section was determined under a microscope. Serum alanine aminotransferase (ALT) levels were significantly lower in the rats injected with LPS and Lipo PGE1 compared with those in the rats injected with LPS alone, and this difference was expressed in a PGE1 dose-dependent manner. Serum MIP-2 levels were significantly lower at 3 h (141.4±95.5 pg/ml) and 8 h (44.9±44.7 pg/ml) after injection with LPS and Lipo-PGE1 (2 μg/kg) than at the same times after injection with LPS alone (342.9±35.9 and 358.3±23.4 pg/ml, respectively). Similarly, serum CINC levels were significantly lower at 8 h (482.7±156.0 ng/ml) after injection with LPS and Lipo-PGE1 (2 μg/kg) than at the same time after injection LPS alone (723.3±29.0 ng/ml). No significant differences were observed at any time between serum tumor necrosis factor-α (TNF-α) levels in rats injected with LPS alone and in rats injected with LPS and Lipo-PGE1 (2 μg/kg). The number of neutrophils that had infiltrated the liver was significantly lower at 8 h after injection with LPS and Lipo PGE1 than at the same time after injection with LPS alone. This difference was expressed in a Lipo PGE1 dose-dependent manner. In conclusion, Lipo PGE1 reduces liver injury and serum levels of MIP-2 and CINC, but not TNF-α, in rats injected with LPS and also reduces the number of neutrophils that infiltrate in the liver.

Pulmonary Protection in Heat Stressed Rats is Associated with Macrophage Inflammatory Protein-2 (MIP-2) Production by Alveolar Macrophages • 201

We investigated whether heat stress protects the lung and cardiovascular system from injury by modifying cytokine production. Methods: 16 rats were randomly assigned to the normothermia group(NG) (n=8) or heat stress group (HSG)(n=8). The HSG maintained a temperature of 42°C for 10 min by external heat. 20 hrs later, the rats were ventilated and given 0.5 mg/kg E. Coli LPS. Blood was collected for blood gases, TNF, IL-10 and MIP-2 at 0, 2, 4, 5 hrs. At 5 hrs, alveolar macrophages (AM) were obtained and incubated for 24 hrs and production of heat shock protein (HSP)was determined. Results: Only the AM of HSG rats produced HSP. By 5 hrs, HSG had a lower A-a O2 gradient compared to the NG rats (209±13 vs 294±22 mm Hg, p<0.01). AM from the HSG produced more MIP-2 (1723±288 vs 751±264 pg/ml, p<0.01). The A-a O2 gradient inversely correlated with MIP-2 in the AM supernatant (r=-0.65, p<0.01, Fig). Hypotension was less in the HSG between 0.5 and 2 hrs but not subsequently. Serum MIP-2 was elevated in the HSG compared to NGat 2 hrs (89.8±32.8 vs 26.5±4.0ng/ml, p<0.02). Detection of TNF, IL-10, and NO in the blood and AM supernatant was similar in both groups. Conclusions: Heat stress-induced lung protection in acute endotoxemia is associated with MIP-2 production by alveolar macrophages. Mechanism of protection from endotoxemia via MIP-2 in heat stressed rats needs to be explored.