Attenuation of retinal endothelial cell migration and capillary morphogenesis in the absence of bcl-2

Apoptosis plays a critical role during development and in the maintenance of the vascular system. B-cell leukemia lymphoma 2 (bcl-2) protects endothelial cells (EC) from apoptosis in response to a variety of stimuli. Previous work from this laboratory demonstrated attenuation of postnatal retinal vascular development and retinal neovascularization during oxygen-induced ischemic retinopathy in bcl-2-deficient (bcl-2-/-) mice. To gain further insight into the function of bcl-2 in the endothelium, we isolated retinal EC from bcl-2+/+ and bcl-2-/- mice. Retinal EC lacking bcl-2 demonstrated reduced cell migration, tenascin-C expression, and adhesion to vitronectin and fibronectin. The bcl-2-/- retinal EC also failed to undergo capillary morphogenesis in Matrigel. In addition, using an ex vivo angiogenesis assay, we observed reduced sprouting from aortic rings grown in culture from bcl-2-/- mice compared with bcl-2+/+ mice. Furthermore, reexpression of bcl-2 was sufficient to restore migration and capillary morphogenesis defects observed in bcl-2-/- retinal EC. Mechanistically, bcl-2-/- cells expressed significantly less endothelial nitric oxide synthase, an important downstream effecter of proangiogenic signaling. This may be attributed to increased oxidative stress in the absence of bcl-2. In fact, incubation of retinal EC or aortic rings from bcl-2-/- mice with the antioxidant N-acetylcysteine rescued their capillary morphogenesis and sprouting defects. Thus, bcl-2-mediated cellular functions play important roles not only in survival but also in proangiogenic phenotype of EC with a significant impact on vascular development and angiogenesis.     

Heme oxygenase-1 induction contributes to renoprotection by G-CSF during rhabdomyolysis-associated acute kidney injury

Granulocyte colony-stimulating factor (G-CSF) is renoprotective during acute kidney injury (AKI) induced by ischemia and cisplatin nephrotoxicity; however, the underlying mechanism is not entirely clear. Rhabdomyolysis is another important clinical cause of AKI, due to the release of nephrotoxins (e.g., heme) from disrupted muscles. The current study has determined the effects of G-CSF on rhabdomyolysis-associated AKI using in vivo and in vitro models. In C57BL/6 mice, intramuscular injection of glycerol induced AKI, which was partially prevented by G-CSF pretreatment. Consistently, glycerol-induced renal tissue damage was ameliorated by G-CSF. In addition, animal survival following the glycerol injection was improved from ～30 to ～70% by G-CSF. In cultured renal tubular cells, hemin-induced apoptosis was also suppressed by G-CSF. Interestingly, G-CSF induced heme oxygenase-1 (HO-1, a critical enzyme for heme/hemin degradation and detoxification) in both cultured tubular cells and mouse kidneys. Blockade of HO-1 with protoporphyrin IX zinc(II) (ZnPP) could largely diminish the protective effects of G-CSF. Together, these results demonstrated the renoprotective effects of G-CSF in rhabdomyolysis-associated AKI. Notably, G-CSF may directly protect against tubular cell injury under the disease condition by inducing HO-1.     

IGF-I deficient mice show reduced peripheral nerve conduction velocities and decreased axonal diameters and respond to exogenous IGF-I treatment

Although insulin-like growth factor-I (IGF-I) can act as a neurotrophic factor for peripheral neurons in vitro and in vivo following injury, the role IGF-I plays during normal development and functioning of the peripheral nervous system is unclear. Here, we report that transgenic mice with reduced levels (two genotypes: heterozygous Igf1+/- or homozygous insertional mutant Igf1m/m) or totally lacking IGF-I (homozygous Igf1-/-) show a decrease in motor and sensory nerve conduction velocities in vivo. In addition, A-fiber responses in isolated peroneal nerves from Igf1+/- and Igf1-/- mice are impaired. The nerve function impairment is most profound in Igf1-/- mice. Histopathology of the peroneal nerves in Igf1-/- mice demonstrates a shift to smaller axonal diameters but maintains the same total number of myelinated fibers as Igf1+/+ mice. Comparisons of myelin thickness with axonal diameter indicate that there is no significant reduction in peripheral nerve myelination in IGF-I-deficient mice. In addition, in Igf1m/m mice with very low serum levels of IGF-I, replacement therapy with exogenous recombinant hIGF-I restores both motor and sensory nerve conduction velocities. These findings demonstrate not only that IGF-I serves an important role in the growth and development of the peripheral nervous system, but also that systemic IGF-I treatment can enhance nerve function in IGF-I-deficient adult mice.     

Alleviation of apoptosis of bone marrow-derived mesenchymal stem cells in the acute injured kidney by heme oxygenase-1 gene modification

Bone marrow-derived mesenchymal stem cells (BMSCs) transplantation is beneficial for the treatment of acute kidney injury (AKI), but the poor survival of BMSCs limits the repair effect. The oxidative stress in the AKI microenvironment is regarded as the main reason. Considering the potent anti-oxidant ability of heme oxygenase-1 (HO-1), HO-1 overexpression in BMSCs can be expected to improve the survival of BMSCs and correspondingly enhance the AKI repair effect. Here, BMSCs are transfected with pLV-HO-1/eGFP and pLV–eGFP by the lentivirus vector to get HO-1-BMSCs and eGFP-BMSCs, respectively. Ischemia/reperfusion-AKI kidney homogenate supernatant (KHS) is prepared for treating BMSCs, eGFP-BMSCs and HO-1-BMSCs. AKI-KHS results in a high inhibitory rate of BMSCs growth and a high proportion of TUNEL positive BMSCs, while HO-1 overexpression inverses this phenomenon and re-establishes the antioxidant and oxidant balance in HO-1-BMSCs. Phosphorylations of p53 and p38 mitogen-activated protein kinases (p38 MAPK) in HO-1-BMSCs decrease. Lower levels of monocyte chemotactic protein 1, tumor necrosis factor-α and interleukin 1β are also observed in supernatant of HO-1-BMSCs. The in vivo study shows that HO-1 overexpression sharply decreases the apoptosis of BMSCs in the injured kidney, and correspondingly the renal function of the AKI rats improves significantly. In conclusion, BMSCs with HO-1 overexpression suggests a better survival in the I/R-AKI microenvironment and a better kidney repair effect. The anti-oxidant effect via the inactivations of the downstream p53 and p38MAPK in BMSCs and the anti-inflammation could be the mechanisms. It provides a novel approach for the cell-based AKI-therapy.     

VEGF-modified human embryonic mesenchymal stem cell implantation enhances protection against cisplatin-induced acute kidney injury

The implantation of mesenchymal stem cells (MSC) has been reported as a new technique to restore renal tubular structure and improve renal function in acute kidney injury (AKI). Vascular endothelial growth factor (VEGF) plays an important role in the renoprotective function of MSC. Whether upregulation of VEGF by a combination of MSC and VEGF gene transfer could enhance the protective effect of MSC in AKI is not clear. We investigated the effects of VEGF-modified human embryonic MSC (VEGF-hMSC) in healing cisplatin-injured renal tubular epithelial cells (TCMK-1) with a coculture system. We found that TCMK-1 viability declined 3 days after cisplatin pretreatment and that coculture with VEGF-hMSC enhanced cell protection via mitogenic and antiapoptotic actions. In addition, administration of VEGF-hMSC in a nude mouse model of cisplatin-induced kidney injury offered better protective effects on renal function, tubular structure, and survival as represented by increased cell proliferation, decreased cellular apoptosis, and improved peritubular capillary density. These data suggest that VEGF-modified hMSC implantation could provide advanced benefits in the protection against AKI by increasing antiapoptosis effects and improving microcirculation and cell proliferation.     

Lipocalin 2 enhances mesenchymal stem cell-based cell therapy in acute kidney injury rat model

Acute kidney injury (AKI) is one of the most common health-threatening diseases in the world. There is still no effective medical treatment for AKI. Recently, Mesenchymal stem cell (MSC)-based therapy has been proposed for treatment of AKI. However, the microenvironment of damaged kidney tissue is not favorable for survival of MSCs which would be used for therapeutic intervention. In this study, we genetically manipulated MSCs to up-regulate lipocalin-2 (Lcn2) and investigated whether the engineered MSCs (MSC-Lcn2) could improve cisplatin-induced AKI in a rat model. Our results revealed that up-regulation of Lcn2 in MSCs efficiently enhanced renal function. MSC Lcn2 up-regulates expression of HGF, IGF, FGF and VEGF growth factors. In addition, they reduced molecular biomarkers of kidney injury such as KIM-1 and Cystatin C, while increased the markers of proximal tubular epithelium such as AQP-1 and CK18 following cisplatin-induced AKI. Overall, here we over-expressed Lcn2, a well-known cytoprotective factor against acute ischemic renal injury, in MSCs. This not only potentiated beneficial roles of MSCs for cell therapy purposes but also suggested a new modality for treatment of AKI.     

Influence of the adenosine A1 receptor on blood pressure regulation and renin release

The present study was performed to investigate the role of adenosine A1 receptors in regulating blood pressure in conscious mice. Adenosine A1-receptor knockout (A1R-/-) mice and their wild-type (A1R+/+) littermates were placed on standardized normal-salt (NS), high-salt (HS), or salt-deficient (SD) diets for a minimum of 10 days before telemetric blood pressure and urinary excretion measurements in metabolic cages. On the NS diet, daytime and nighttime mean arterial blood pressure (MAP) was 7-10 mmHg higher in A1R-/- than in A1R+/+ mice. HS diet did not affect the MAP in A1R-/- mice, but the daytime and nighttime MAP of the A1R+/+ mice increased by approximately 10 mmHg, to the same level as that in the A1R-/-. On the SD diet, day- and nighttime MAP decreased by approximately 6 mmHg in both A1R-/- and A1R+/+ mice, although the MAP remained higher in A1R-/- than in A1R+/+ mice. Although plasma renin levels decreased with increased salt intake in both genotypes, the A1R-/- mice had an approximately twofold higher plasma renin concentration on all diets compared with A1R+/+ mice. Sodium excretion was elevated in the A1R-/- compared with the A1R+/+ mice on the NS diet. There was no difference in sodium excretion between the two genotypes on the HS diet. Even on the SD diet, A1R-/- mice had an increased sodium excretion compared with A1R+/+ mice. An abolished tubuloglomerular feedback response and reduced tubular reabsorption can account for the elevated salt excretion found in A1R-/- animals. The elevated plasma renin concentrations found in the A1R-/- mice could also result in increased blood pressure. Our results confirm that adenosine, acting through the adenosine A1 receptor, plays an important role in regulating blood pressure, renin release, and sodium excretion.     

Myeloid heme oxygenase-1 haploinsufficiency reduces high fat diet-induced insulin resistance by affecting adipose macrophage infiltration in mice

Increased adipose tissue macrophages contribute to obesity-induced metabolic syndrome. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with potent anti-inflammatory and proangiogenic activities in macrophages. However, the role of macrophage HO-1 on obesity-induced adipose inflammation and metabolic syndrome remains unclear. Here we show that high-fat diet (HFD) feeding in C57BL/6J mice induced HO-1 expression in the visceral adipose tissue, particularly the stromal vascular fraction. When the irradiated C57BL/6J mice reconstituted with wild-type or HO-1(+/-) bone marrow were fed with HFD for over 24 weeks, the HO-1(+/-) chimeras were protected from HFD-induced insulin resistance and this was associated with reduced adipose macrophage infiltration and angiogenesis, suggesting that HO-1 affects myeloid cell migration toward adipose tissue during obesity. In vivo and in vitro migration assays revealed that HO-1(+/-) macrophages exhibited an impaired migration response. Chemoattractant-induced phosphorylation of p38 and focal adhesion kinase (FAK) declined faster in HO-1(+/-) macrophages. Further experiments demonstrated that carbon monoxide and bilirubin, the byproducts derived from heme degradation by HO-1, enhanced macrophage migration by increasing phosphorylation of p38 and FAK, respectively. These data disclose a novel role of hematopoietic cell HO-1 in promoting adipose macrophage infiltration and the development of insulin resistance during obesity.     

Role of ICAM-1 (CD54) in the development of murine cerebral malaria

In susceptible mouse strains, infection of mice with Plasmodium berghei ANKA (PbA) results in a lethal complication, cerebral malaria. Cerebral malaria is due to the immune response induced by the parasite, which results in an increased production of TNF, known to increase the expression of adhesion molecules on the endothelia. To investigate the role of the adhesion molecule ICAM-1 (CD54), we infected wild-type (+/+) and ICAM-1-deficient (-/-) mice with PbA. While +/+ mice died 6-8 days after infection, -/- mice survived > 15 days. Parasitaemia was similar in +/+ and -/- mice. Serum TNF concentration was increased by the infection and was significantly higher in infected +/+ than in -/- mice. However, TNF mRNA levels in spleen, lungs, and brain were elevated in both infected +/+ and -/- mice. For IFN-gamma, serum levels were similar in both groups. A breakdown of the blood-brain barrier was evident in infected +/+ mice only. Interestingly, thrombocytopenia was profound in infected +/+, but practically absent in -/- mice. Moreover, macrophage sequestration was evident in brain venules and lung capillaries of +/+ mice and was significantly less important in the alveolar capillaries of infected -/- mice. In contrast, neutrophil sequestration in the lung was similar in both +/+ and -/- mice. Sequestration of parasitized red blood cells was significantly greater in the alveolar capillaries from +/+ than -/- mice. These results indicate that while the immune response is similar in both +/+ and ICAM-1(-/-) mice, the absence of mortality in ICAM(-/-) mice correlates with a decrease of macrophage and parasitized RBC trapping and a less severe thrombocytopenia.     

CC chemokine receptor 1 enhances susceptibility to Leishmania major during early phase of infection

CC chemokine receptor 1 (CCR1) is expressed on the surfaces of monocytes, lymphocytes, neutrophils and eosinophils. CC chemokine receptor 1 not only regulates leucocyte chemotaxis, but also plays a role in the regulation of Th1/Th2 cytokine responses. To determine the role of CCR1 in regulation of immune response during Leishmania major infection, we analysed the course of cutaneous L. major infection in CCR1-deficient C57BL/6 mice (CCR1-/-) and compared with similarly infected wild-type mice (CCR1+/+). Following L. major infection, CCR1-/- mice developed significantly smaller lesions containing fewer parasites than CCR1+/+ mice. Furthermore, the severity of the inflammation as assessed by the degree of leucocyte infiltration at the site of infection was similar in CCR1+/+ and CCR1-/- mice. Although both groups developed significant antibody responses following L. major infection, CCR1-/- mice produced significantly lower IgE. On day 20 postinfection, LmAg-stimulated lymph node cells from L. major-infected CCR1+/+ and CCR1-/- mice produced comparable levels of IL-12 and IFN-gamma, but those from CCR1-/- mice produced significantly less IL-4 and IL-10. By day 70, lymph node cells from both CCR1+/+ and CCR1-/- mice produced significant amounts of IL-12 and IFN-gamma but low IL-4. At both time points, the draining lymph nodes from CCR1+/+ and CCR1-/- mice contained similar number of leucocytes. These results demonstrate that CCR1 plays a role in pathogenesis of cutaneous L. major infection. Moreover, they also indicate that CCR1 exacerbates L. major infection in C57BL/6 mice by up-regulating Th2-like response rather than inhibiting Th1 development or/and influencing leucocyte chemotaxis.     

Loss of ACE2 Exacerbates Murine Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion (I/R) is a model of acute kidney injury (AKI) that is characterized by vasoconstriction, oxidative stress, apoptosis and inflammation. Previous studies have shown that activation of the renin-angiotensin system (RAS) may contribute to these processes. Angiotensin converting enzyme 2 (ACE2) metabolizes angiotensin II (Ang II) to angiotensin-(1–7), and recent studies support a beneficial role for ACE2 in models of chronic kidney disease. However, the role of ACE2 in models of AKI has not been fully elucidated. In order to test the hypothesis that ACE2 plays a protective role in AKI we assessed I/R injury in wild-type (WT) mice and ACE2 knock-out (ACE2 KO) mice. ACE2 KO and WT mice exhibited similar histologic injury scores and measures of kidney function at 48 hours after reperfusion. Loss of ACE2 was associated with increased neutrophil, macrophage, and T cell infiltration in the kidney. mRNA levels for pro-inflammatory cytokines, interleukin-1β, interleukin-6 and tumour necrosis factor-α, as well as chemokines macrophage inflammatory protein 2 and monocyte chemoattractant protein-1, were increased in ACE2 KO mice compared to WT mice. Changes in inflammatory cell infiltrates and cytokine expression were also associated with greater apoptosis and oxidative stress in ACE2 KO mice compared to WT mice. These data demonstrate a protective effect of ACE2 in I/R AKI.     

Protective roles of mast cells and mast cell-derived TNF in murine malaria

TNF plays important roles in the protection and onset of malaria. Although mast cells are known as a source of TNF, little is known about the relationship between mast cells and pathogenesis of malaria. In this study, mast cell-deficient WBB6F1-W/W(v) (W/W(v)) and the control littermate WBB6F1+/+ (+/+) mice were infected with 1 x 10(5) of Plasmodium berghei ANKA. +/+ mice had lower parasitemia with higher TNF levels, as compared with W/W(v) mice. Diminished resistance in W/W(v) mice was considered to be due to mast cells and TNF. This fact was confirmed by experiments in W/W(v) mice reconstituted with bone marrow-derived mast cells (BMMCs) of +/+ mice or of TNF-/- mice. W/W(v) mice with BMMCs of +/+ mice exhibit lower parasitemia and mortality accompanying significantly higher TNF levels than those of W/W(v) mice. Parasitemia in W/W(v) mice with BMMCs of TNF-/- mice was higher than that in +/+ mice. Activation of mast cells by anti-IgE or compound 48/80 resulted in release of TNF and decrease of parasitemia. In addition, splenic hypertrophy and increased number of mast cells in the spleen were observed after infection in +/+ mice and W/W(v) mice reconstituted with BMMCs of +/+ mice as compared with W/W(v) mice. These findings propose a novel mechanism that mast cells and mast cell-derived TNF play protective roles in malaria.     

Role of CC chemokine receptor 2 in bone marrow cells in the recruitment of macrophages into obese adipose tissue

The MCP-1 (monocyte chemoattractant protein-1)/CCR2 (CC motif chemokine receptor-2) pathway may play a role in macrophage infiltration into obese adipose tissue. Here we investigated the role of CCR2 in the recruitment of bone marrow-derived macrophages into obese adipose tissue in vitro and in vivo. Using the TAXIScan device, which can measure quantitatively the directionality and velocity of cell migration at time lapse intervals in vitro, we demonstrated that bone marrow cells (BMCs) from wild type mice migrate directly toward MCP-1 or culture medium conditioned by adipose tissue explants of genetically obese ob/ob mice, which are efficiently suppressed by pharmacological blockade of CCR2 signaling. The number of F4/80-positive macrophages was reduced in the adipose tissue from high fat diet-fed obese KKAy or ob/ob mice treated with a CCR2 antagonist propagermanium relative to vehicle-treated groups. We also found that the number of macrophages is reduced in the adipose tissue from ob/ob mice reconstituted with CCR2(-/-) BMCs (ob/ob + CCR2(-/-) BMCs) relative to those with CCR2+/+ BMCs (ob/ob + CCR2+/+ BMCs). Expression of mRNAs for CD11c and TLR4 (Toll-like receptor 4) markers of proinflammatory M1 macrophages was also decreased in the adipose tissue from ob/ob + CCR2(-/-) BMCs relative to ob/ob + CCR2+/+ BMCs, whereas mannose receptor and CD163, markers of anti-inflammatory M2 macrophages, were unchanged. This study provides in vivo and in vitro evidence that CCR2 in bone marrow cells plays an important role in the recruitment of macrophages into obese adipose tissue.     

Genetically resistant mice lacking IL-18 gene develop Th1 response and control cutaneous Leishmania major infection

IL-18 has been shown to play a critical role in the development of a Th1 response and immunity against intracellular pathogens. To determine the role of IL-18 in the development of protective immunity against Leishmania major, we have analyzed the course of cutaneous L. major in IL-18-deficient C57BL/6 mice (IL-18-/-) compared with similarly infected wild-type mice (IL-18+/+). After L. major infection, IL-18-/- mice may develop larger lesions during early phase of infection but eventually will resolve them as efficiently as IL-18+/+ mice. By 2 wk after infection, although Ag-stimulated lymph node cells from L. major-infected IL-18+/+ and IL-18-/- mice produced similar levels of IFN-gamma, those from IL-18-/- mice produced significantly more IL-12 and IL-4. By 10 wk after infection, both IL-18+/+ and IL-18-/- mice had resolved L. major infection. At this time, lymph node cells from both IL-18+/+ and IL-18-/- mice produced IL-12 and IFN-gamma but no IL-4. Furthermore, administration of anti-IFN-gamma Abs to IL-18-/- mice rendered them susceptible to L. major. These results indicate that despite the role IL-18 may play in early control of cutaneous L. major lesion growth, this cytokine is not critical for development of protective Th1 response and resolution of L. major infection.     

Superoxide dismutase 1 protects retinal cells from oxidative damage

Bolstering the endogenous oxidative damage defense system is a good strategy for development of treatments to combat neurodegenerative diseases in which oxidative damage plays a role. A first step in such treatment development is to determine the role of various components of the defense system in cells that degenerate. In this study, we sought to determine the role of superoxide dismutase 1 (SOD1) in two models of oxidative damage-induced retinal degeneration. In one model, paraquat is injected into the vitreous cavity and then enters retinal cells and generates reactive oxygen species (ROS) that cause progressive retinal damage. Assessment of retinal function with serial electroretinograms (ERGs) showed that sod1 -/- mice were much more sensitive than sod1 +/+ mice to the damaging effects of paraquat, while sod1 +/- mice showed intermediate sensitivity. Compared to sod1 +/+ mice, sod1 -/- mice showed greater paraquat-induced oxidative damage and apoptosis. In the second model, mice were exposed to hyperoxia for several weeks, and sod1 -/- mice showed significantly greater reductions in ERG amplitudes than sod1 +/+ mice. In both of these models, transgenic mice carrying a sod1 transgene driven by a beta-actin promoter showed less oxidative stress-induced reduction in ERG amplitudes. These data demonstrate that SOD1 protects retinal cells against paraquat- and hyperoxia-induced oxidative damage and suggest that overexpression of SOD1 should be considered as one component of ocular gene therapy to prevent oxidative damage-induced retinal degeneration.     

Important role of apoptosis signal-regulating kinase 1 in ischemic acute kidney injury

We investigated the role of apoptosis signal-regulating kinase 1 (ASK1) in ischemia/reperfusion (I/R)-induced acute kidney injury (AKI). Blood urea nitrogen (BUN) and serum creatinine were significantly higher in ASK1+/+ mice than in ASK1−/− mice after I/R injury. Renal histology of ASK1+/+ mice showed significantly greater tubular necrosis and degradation. In ASK1−/− mice, phosphorylation of ASK1, JNK, and p38K, and the number of TUNEL-positive cells and infiltrated leukocytes decreased after I/R injury. Apoptotic changes were significantly decreased in cultured renal tubular epithelial cells (TECs) from ASK1−/− mice under hypoxic condition. Transfection with dominant-active ASK1 induced apoptosis in TECs. Protein expression of monocyte chemoattractant protein-1 (MCP-1) was significantly weaker in ASK1−/− mice after I/R injury. Transfection with dominant negative-ASK1 significantly decreased MCP-1 production in TECs. These results demonstrated that ASK1 is activated in I/R-induced AKI, and blockage of ASK1 attenuates renal tubular apoptosis, MCP-1 expression, and renal function.     

In vivo evidence for the role of GM-CSF as a mediator in acute pancreatitis-associated lung injury

Severe pancreatitis is frequently associated with acute lung injury (ALI) and the respiratory distress syndrome. The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in mediating the ALI associated with secretagogue-induced experimental pancreatitis was evaluated with GM-CSF knockout mice (GM-CSF -/-). Pancreatitis was induced by hourly (12x) intraperitoneal injection of a supramaximally stimulating dose of the cholecystokinin analog caerulein. The resulting pancreatitis was similar in GM-CSF-sufficient (GM-CSF +/+) control animals and GM-CSF -/- mice. Lung injury, quantitated by measuring lung myeloperoxidase activity (an indicator of neutrophil sequestration), alveolar-capillary permeability, and alveolar membrane thickness was less severe in GM-CSF -/- than in GM-CSF +/+ mice. In GM-CSF +/+ mice, pancreas, lung and serum GM-CSF levels increase during pancreatitis. Lung levels of macrophage inflammatory protein (MIP)-2 are also increased during pancreatitis, but, in this case, the rise is less profound in GM-CSF -/- mice than in GM-CSF +/+ controls. Administration of anti-MIP-2 antibodies was found to reduce the severity of pancreatitis-associated ALI. Our findings indicate that GM-CSF plays a critical role in coupling pancreatitis to ALI and suggest that GM-CSF may act indirectly by regulating the release of other proinflammatory factors including MIP-2.