Macrophage inflammatory protein-1

Macrophage inflammatory protein-1 (MIP-1) and MIP-1β are highly related members of the CC chemokine subfamily. Despite their structural similarities, MIP-1 and MIP-1β show diverging signaling capacities. Depending on the MIP-1 subtype and its NH₂-terminal processing, one or more of the CC chemokine receptors CCR1, CCR2, CCR3 and CCR5 are recognized. Since both human MIP-1 subtypes (LD78 and LD78β) and MIP-1β signal through CCR5, the major co-receptor for M-tropic HIV-1 strains, these chemokines are capable of inhibiting HIV-1 infection in susceptible cells. In this review, different aspects of human and mouse MIP-1 and MIP-1β are discussed, including their protein and gene structures, their regulated production, their receptor usage and biological activities and their role in several pathologies including HIV-1 infection.     

Macrophage inflammatory protein-1

Macrophage inflammatory protein (MIP)-1alpha was identified 15 years ago as the first of now four members of the MIP-1 CC chemokine subfamily. These proteins termed CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CCL9/10 (MIP-1delta), and CCL15 (MIP-1gamma) according to the revised nomenclature for chemokines are produced by many cells, particularly macrophages, dendritic cells, and lymphocytes. MIP-1 proteins, which act via G-protein-coupled cell surface receptors (CCR1, 3, 5), e.g. expressed by lymphocytes and monocytes/macrophages (MPhi), are best known for their chemotactic and proinflammatory effects but can also promote homoeostasis. The encouraging results of preclinical studies in murine models of inflammation, i.e. asthma, arthritis, or multiple sclerosis, have led to the development of potent CCR3 and 5 antagonists, some of which are currently being tested in first clinical trials.     

Macrophage inflammatory protein-2 and vascular endothelial growth factor regulate corneal neovascularization induced by infection with Pseudomonas aeruginosa in mice

Pseudomonas aeruginosa ocular infection causes extensive corneal neovascularization. The purpose of the present study was to investigate the role of the angiogenic factors macrophage inflammatory protein-2 (MIP-2) and vascular endothelial growth factor (VEGF) in the regulation of corneal neovascularization during P. aeruginosa ocular infection. After administering anti-MIP-2 antibody or control antibody, mouse corneas were challenged with P. aeruginosa. The expression of MIP-2 and VEGF was detected using an ELISA from ocular homogenates. Corneal neovascularization was examined by histology. The cellular sources of MIP-2 and VEGF were identified by immunohistochemistry. In addition, protein expression of MIP-2 and VEGF in isolated corneas was measured to determine the ability of the cornea to produce these two mediators. Results showed that the expression of MIP-2 and VEGF was significantly (P < 0.05) elevated after bacterial infection, and high levels of these two mediators paralleled the extensive corneal neovascularization seen at later stages of the infection. Anti-MIP-2 antibody treatment resulted in a significant (P < 0.05) reduction in VEGF expression and in corneal neovascularization. Both corneal resident cells and infiltrating neutrophils had the ability to produce MIP-2 and VEGF after stimulation. The present study demonstrates that both MIP-2 and VEGF are important mediators in the regulation of corneal neovascularization caused by P. aeruginosa infection, and that MIP-2 regulates the production of VEGF.     

Macrophage migration inhibitory factor promotes renal injury induced by ischemic reperfusion

Macrophage migration inhibitory factor (MIF) is pleiotropic cytokine that has multiple effects in many inflammatory and immune diseases. This study reveals a potential role of MIF in acute kidney injury (AKI) in patients and in kidney ischemic reperfusion injury (IRI) mouse model in MIF wild‐type (WT) and MIF knockout (KO) mice. Clinically, plasma and urinary MIF levels were largely elevated at the onset of AKI, declined to normal levels when AKI was resolved and correlated tightly with serum creatinine independent of disease causes. Experimentally, MIF levels in plasma and urine were rapidly elevated after IRI‐AKI and associated with the elevation of serum creatinine and the severity of tubular necrosis, which were suppressed in MIF KO mice. It was possible that MIF may mediate AKI via CD74/TLR4‐NF‐κB signalling as mice lacking MIF were protected from AKI by largely suppressing CD74/TLR‐4‐NF‐κB associated renal inflammation, including the expression of MCP‐1, TNF‐α, IL‐1β, IL‐6, iNOS, CXCL15(IL‐8 in human) and infiltration of macrophages, neutrophil, and T cells. In conclusion, our study suggests that MIF may be pathogenic in AKI and levels of plasma and urinary MIF may correlate with the progression and regression of AKI.     

Macrophage migration inhibitory factor mediates late cardiac dysfunction after burn injury

We have recently demonstrated that macrophage migration inhibitory factor (MIF) is a myocardial depressant protein and that MIF mediates late, prolonged cardiac dysfunction after endotoxin challenge in mice. Because many factors, including endotoxin, have been implicated in the pathogenesis of cardiac dysfunction after burn injury, we tested the hypothesis that MIF might also be the mediator of prolonged cardiac dysfunction in this model. At 4 h after 40% total body surface area burn in anesthetized mice, serum MIF levels increased significantly compared with baseline (2.2-fold). This increase was accompanied by a significant decrease in cardiac tissue MIF levels (2.1-fold decrease compared with controls). This pattern was consistent with MIF release from preformed cytoplasmic stores in the heart and other organs. To determine whether MIF mediates cardiac dysfunction after burn injury, mice were pretreated with anti-MIF neutralizing monoclonal antibodies or isotype control antibodies. Beginning 4 h after burn injury (and continuing through 48 h), burned mice demonstrated a significantly depressed left ventricular shortening fraction of 38.6 +/- 1.8%, compared with the normal controls (56.0 +/- 2.6%). Mice treated with anti-MIF displayed an initial depression of cardiac function similar to nontreated animals but then showed rapid restoration of cardiac function with complete recovery by 24 h, which persisted for the duration of the protocol. This study is the first to demonstrate that MIF mediates late, prolonged cardiac dysfunction after burn injury and suggests that MIF blockade should be considered a therapeutic target for the treatment of burn injury.     

Profile of gastrointestinal damage induced by platelet-activating factor

The ulcerogenic actions of an intravenous infusion of platelet-activating factor (100 ng/kg/min) was studied in the rat. Damage to the stomach, duodenum, jejunum, ileum and colon were assessed histologically and using intraluminal acid phosphatase release as a marker of cellular damage. A 10-min infusion of platelet-activating factor caused extensive haemorrhagic damage to each of the regions examined, with the exception of the colon. Acid phosphatase release was significantly elevated in the stomach, jejunum, ileum (p less than 0.001) and duodenum (p less than 0.01), but not in the colon. These studies demonstrate that platelet-activating factor is a potent ulcerogen in the stomach and small intestine, and support a role for this endogenous phospholipid as a mediator of the ulceration associated with endotoxin-induced shock.     

Profile of gastrointestinal damage induced by platelet-activating factor

The ulcerogenic actions of an intravenous infusion of platelet-activating factor (100 ng/kg/min) was studied in the rat. Damage to the stomach, duodenum, jejunum, ileum and colon were assessed histologically and using intraluminal acid phosphatase release as a marker of cellular damage. A 10-min infusion of platelet-activating factor caused extensive haemorrhagic damage to each of the regions examined, with the exception of the colon. Acid phosphatase release was significantly elevated in the stomach, jejunum, ileum (p<0.001) and duodenum (p<0.01), but not in the colon. These studies demonstrate that platelet-activating factor is a potent ulcerogen in the stomach and small intestine, and support a role for this endogenous phospholipid as a mediator of the ulceration associated with endotoxin-induced shock.     

Zinc acexamate reduces gastric damage induced by platelet-activating factor

We have tested the ability of zinc acexamate (ZAC) to prevent platelet-activating-factor (Paf) induced gastric damage in rats. Lesions were characterized by a vascular congestion affecting the entire mucosa, oedema, haemorrhage and frequent necrosis of the more superficial areas. The gastric damage appearing after Paf was accompanied by degranulation of gastric mast cells. Leukocytes were often seen at the submucosal level. Oral pretreatment with ZAC reduced in a dose-dependent manner both gastric damage and mast cell degranulation observed after Paf. ZAC administered orally at a dose of 100 mg kg-1 statistically inhibited (p less than 0.01) gastric damage and mast cell degranulation. ZAC did not affect the hypotension induced by Paf confirming that gastric damage and hypotension appearing in rats after Paf administration are two independent phenomena. The present findings indicate that the inhibitory effect of ZAC upon gastric lesions induced by Paf may be related to the different protective actions exhibited by this zinc compound in a wide variety of experimental models of gastric ulcer.     

Macrophage migration inhibitory factor mediates hypoxia-induced pulmonary hypertension

Pulmonary hypertension (PH) is a devastating disease leading to progressive hypoxemia, right ventricular failure, and death. Hypoxia can play a pivotal role in PH etiology, inducing pulmonary vessel constriction and remodeling. These events lead to increased pulmonary vessel wall thickness, elevated vascular resistance and right ventricular hypertrophy. The current study examined the association of the inflammatory cytokine macrophage migration inhibitory factor (MIF) with chronic lung disease and its role in the development of hypoxia-induced PH. We found that plasma MIF in patients with primary PH or PH secondary to interstitial lung disease (ILD) was significantly higher than in the control group (P = 0.004 and 0.007, respectively). MIF involvement with hypoxia-induced fibroblast proliferation was examined in both a human cell-line and primary mouse cells from wild-type (mif ^+/+) and MIF-knockout (mif ^−/−) mice. In vitro, hypoxia-increased MIF mRNA, extracellular MIF protein accumulation and cell proliferation. Inhibition of MIF inflammatory activity reduced hypoxia-induced cell proliferation. However, hypoxia only increased proliferation of mif ^−/− cells when they were supplemented with media from mif ^+/+ cells. This growth increase was suppressed by MIF inhibition. In vivo, chronic exposure of mice to a normobaric atmosphere of 10% oxygen increased lung tissue expression of mRNA encoding MIF and accumulation of MIF in plasma. Inhibition of the MIF inflammatory active site, during hypoxic exposure, significantly reduced pulmonary vascular remodeling, cardiac hypertrophy and right ventricular systolic pressure. The data suggest that MIF plays a critical role in hypoxia-induced PH, and its inhibition may be beneficial in preventing the development and progression of the disease.     

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) E₂ 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 PGE₂ 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.     

Activator protein-1 mediates induced but not basal epidermal growth factor receptor gene expression

BACKGROUND: The epidermal growth factor receptor (EGFR) is expressed at different levels in many cell types and found overexpressed in many cancers. EGFR expression is increased or decreased in response to extracellular stimuli. We examined the effect of increased c-Jun expression on EGFR promoter activity. MATERIALS AND METHODS: We used DNAse I foot-printing analysis to determine the binding of activator protein 1 (AP-1) to the promoter region. We also used cotransfection experiments and western blotting analysis to determine the effect of AP-1 family members on EGFR expression. RESULTS: AP-1 was able to bind to at least seven sites in the EGFR promoter region. Cotransfection of MCF-7 cells with a c-Jun expression vector and the EGFR promoter reporter resulted in a 7-fold increase in promoter activity. JunB, but not c-fos, also enhanced the EGFR promoter activity. An A-Fos-dominant negative shown to inhibit Jun-dependent transactivation was able to prevent c-Jun induction of the promoter activity, but only slightly decreased the basal activity of the promoter. Furthermore, the A-Fos dominant negative was able to inhibit phorbol ester induction of the EGFR promoter. Examination of EGFR expression of MCF-7 stable cell lines that overexpress c-Jun revealed an increase in EGFR expression. Additionally, a cisplatin-resistant cell line, A2780/CP70, which has an increase in AP-1 activity compared with the parental cell line, A2780, was found to have an increase in EGFR level. CONCLUSIONS: These results indicate that AP-1 can act to increase the expression of EGFR and may play a role in upregulation of EGFR in cancer cells.     

Mitochondrial uncoupling protein-2 mediates steatotic liver injury following ischemia/reperfusion

Steatotic livers are not used for transplantation because they have a reduced tolerance for ischemic events with reduced ATP levels and greater levels of cellular necrosis, which ultimately result in total organ failure. Mitochondrial uncoupling protein-2 (UCP2) is highly expressed in steatotic livers and may be responsible for liver sensitivity to ischemia through mitochondrial and ATP regulation. To test this hypothesis, experiments were conducted in lean and steatotic (ob/ob), wild-type, and UCP2 knock-out mice subjected to total warm hepatic ischemi-a/reperfusion. Although ob/ob UCP2 knock-out mice and ob/ob mice have a similar initial phenotype, ob/ob UCP2 knock-out animal survival was 83% when compared with 30% in ob/ob mice 24 h after reperfusion. Serum alanine aminotransferase concentrations and hepatocellular necrosis were decreased in the ob/ob UCP2 knock-out mice when compared with ob/ob mice subjected to ischemia. Liver ATP levels were increased in the ob/ob UCP2 knock-out animals after reperfusion when compared with the ob/ob mice but remained below the concentrations from lean livers. Lipid peroxidation (thiobarbituric acid-reactive substances) increased after reperfusion most significantly in the steatotic groups, but the increase was not affected by UCP2 deficiency. These results reveal that UCP2 expression is a critical factor, which sensitizes steatotic livers to ischemic injury, regulating liver ATP levels after ischemia and reperfusion.     

Macrophage inflammatory protein-1alpha expression plasmid enhances DNA vaccine-induced immune responses against HSV-2

In this study, we examined the effectiveness of macrophage inflammatory protein (MIP)-1alpha cDNA as a HSV-2 DNA vaccine adjuvant. pcDNA3-gD (pgD) and pcDNA3-MIP-1alpha (pMIP-1alpha) were co-injected to examine the modulatory effects of MIP-1alpha on immune phenotype and protection against lethal challenge with HSV-2. We found that Th-cell proliferative responses were dramatically enhanced by co-injection of pgD and pMIP-1alpha compared with injection of pgD alone. The secretion of IL-2 and IFN-gamma was also significantly increased by pgD and pMIP-1alpha co-injection; however, the production of cytokines IL-4 and IL-10 was not affected by co-injection. pgD and pMIP-1alpha co-injection resulted in a moderate enhancement of systemic gD-specific antibody level, but mucosal secretory IgA was markedly enhanced. When BALB/c mice were challenged intravaginally with 100 LD50 of HSV-2 strain Sav, pMIP-1alpha co-injection with pgD improved their survival rate and significantly reduced both the number of mice with lesions and the lesion severity. Therefore, MIP-1alpha cDNA as a HSV-2 DNA vaccine adjuvant drives antigen-specific Th1-type responses, reducing HSV-2-derived morbidity and mortality.     

Macrophage inflammatory protein-2 is a mediator of polymorphonuclear neutrophil influx in ocular bacterial infection

Polymorphonuclear neutrophils (PMN) in Pseudomonas aeruginosa-infected cornea are required to clear bacteria from affected tissue, yet their persistence may contribute to irreversible tissue destruction. This study examined the role of C-X-C chemokines in PMN infiltration into P. aeruginosa-infected cornea and the contribution of these mediators to disease pathology. After P. aeruginosa challenge, corneal PMN number and macrophage inflammatory protein-2 (MIP-2) and KC levels were compared in mice that are susceptible (cornea perforates) or resistant (cornea heals) to P. aeruginosa infection. While corneal PMN myeloperoxidase activity (indicator of PMN number) was similar in both groups of mice at 1 and 3 days postinfection, by 5-7 days postinfection corneas of susceptible mice contained a significantly greater number of inflammatory cells. Corneal MIP-2, but not KC, levels correlated with persistence of PMN in the cornea of susceptible mice. To test the biological relevance of these data, resistant mice were treated systemically with rMIP-2. This treatment resulted in increased corneal PMN number and significantly exacerbated corneal disease. Conversely, administration of neutralizing MIP-2 pAb to susceptible mice reduced both PMN infiltration and corneal destruction. Collectively, these findings support an important role for MIP-2 in recruitment of PMN to P. aeruginosa-infected cornea. These data also strongly suggest that a timely down-regulation of the host inflammatory response is critical for resolution of infection.     

Macrophage inflammatory protein-2 is required for neutrophil passage across the epithelial barrier of the infected urinary tract

IL-8 is a major human neutrophil chemoattractant at mucosal infection sites. This study examined the C-X-C chemokine response to mucosal infection, and, specifically, the role of macrophage inflammatory protein (MIP)-2, one of the mouse IL-8 equivalents, for neutrophil-epithelial interactions. Following intravesical Escherichia coli infection, several C-X-C chemokines were secreted into the urine, but only MIP-2 concentrations correlated to neutrophil numbers. Tissue quantitation demonstrated that kidney MIP-2 production was triggered by infection, and immunohistochemistry identified the kidney epithelium as a main source of MIP-2. Treatment with anti-MIP-2 Ab reduced the urine neutrophil numbers, but the mice had normal tissue neutrophil levels. By immunohistochemistry, the neutrophils were found in aggregates under the pelvic epithelium, but in control mice the neutrophils crossed the urothelium into the urine. The results demonstrate that different chemokines direct neutrophil migration from the bloodstream to the lamina propria and across the epithelium and that MIP-2 serves the latter function. These findings suggest that neutrophils cross epithelial cell barriers in a highly regulated manner in response to chemokines elaborated at this site. This is yet another mechanism that defines the mucosal compartment and differentiates the local from the systemic host response.     

Platelet aggregation induced by platelet-activating factor is suppressed by cystine protease inhibitor

The effect of NCO-700, a cystine protease inhibitor, on platelet-activating factor-induced platelet aggregation was determined. A newly synthesized cystine protease inhibitor (calcium-activated neutral protease and cathepsin B inhibitor), NCO-700 (bis[ethyl (2R, 3R)-3-[(S)-methyl-1-[4-(2,3,4- trimethoxyphenylmethyl) piperazine-1-ylcarbonyl]butylcarbonyl]oxiran-2-carboxy late]sulfate), inhibited platelet-activating factor-induced platelet aggregation. The inhibition was dependent on the preincubation time with NCO-700 and on the concentration of the inhibitor. The release of serotonin was also inhibited almost completely by the 20-min preincubation with 10(-4) M NCO-700. Leupeptin also inhibited platelet-activating factor-induced platelet aggregation. But calcium-activated neutral protease inhibitor did not inhibit it. These observations suggest that NCO-700-sensitive protease(s) such as cystine protease may contribute to platelet aggregation induced by platelet-activating factor.     

Role of thromboxane A2 in airway microvascular leakage induced by inhaled platelet-activating factor.

We studied the effects of OKY-046 (1, 10, and 30 mg/kg iv), a selective thromboxane synthase inhibitor, and of ICI 192605 (0.5 mg/kg), a selective thromboxane A2 receptor antagonist, on airflow obstruction and airway microvascular leakage induced by inhaled platelet-activating factor (PAF). Extravasated Evans blue dye content was measured as a reflection of airway microvascular leakage. In control animals, PAF caused a significantly higher increase in extravasation of dye and significantly less increase in lung resistance (RL) than histamine. OKY-046 significantly inhibited both changes in RL and airway microvascular leakage after PAF in a dose-dependent manner, whereas it inhibited histamine-induced airway microvascular leakage only at main bronchi, without any significant effect on RL. ICI 192605 significantly inhibited both RL and airway microvascular leakage induced by PAF, but not after histamine. After both PAF and histamine, changes in RL correlated significantly with the degree of microvascular leakage. Airway microvascular leakage and airflow obstruction after PAF, but not after histamine, may be dependent on thromboxane A2 generation.     

Macrophage inflammatory protein-2 (MIP-2)/CXCR2 blockade attenuates acute graft-versus-host disease while preserving graft-versus-leukemia activity

Allogenic bone marrow transplantation (BMT), an important treatment for hematological malignancies, is often complicated by graft-versus-host disease (GVHD). Suppression of GVHD is associated with the unwanted diminishment of the graft-versus-leukemia (GVL) response. The aim of this study was to maintain the benefits of GVL during GVHD suppression through isolated blockade of T-cell migration factors. To this end, we developed a murine model of B-cell leukemia, which was treated with BMT to induce GVHD. Within this model, functional blockade of MIP-2/CXCR2 was analyzed by observing proteomic, histologic and clinical variables of GVHD manifestation. Luminex assay of collected tissue identified several cytokines [granulocyte colony-stimulating factor (G-CSF), keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2), and interleukin-23 (IL-23)] that were upregulated during GHVD, but reduced by neutralizing the MIP-2/CXCR2 axis. In addition, donor T-cell blockade of CXCR2 combined with recipient administration of anti-MIP-2 caused a significant decrease in GVHD while preserving the GVL response. We propose that blocking the MIP-2/CXCR2 axis represents a novel strategy to separate the toxicity of GVHD from the beneficial effects of GVL after allogenic BMT.