Expression Of MIP-1alpha Research Articles

CCL2 Inhibition of Pro-Resolving Mediators Potentiates Neuroinflammation in Astrocytes.

The chemokine CCL2 participates in multiple neuroinflammatory processes, mainly through the recruitment of glial cells. However, CCL2 has also been proven to exert different types of actions on these cells, including the modification of their response to inflammatory stimuli. In the present study we analyzed the effect of CCL2 on the resolution of inflammation in astrocytes. We observed that genetic removal of CCL2 increases the expression of the enzymes responsible for the synthesis of specialized pro-resolving mediators arachidonate 15-lipoxygenase and arachidonate 5-lipoxygenase in the brain cortex of 5xFAD mice. The expression of FPR2 receptor, known to mediate the activity of pro-resolving mediators was also increased in mice lacking CCL2.The downregulation of these proteins by CCL2 was also observed in cultured astrocytes. This suggests that CCL2 inhibition of the resolution of inflammation could facilitate the progression of neuroinflammatory processes. The production of the pro-inflammatory cytokine IL-1beta by astrocytes was analyzed, and allowed us to confirm that CCL2 potentiates the activation of astrocytes trough the inhibition of pro-resolving pathways mediated by Resolvin D1. In addition, the analysis of the expression of TNFalpha, MIP1alpha and NOS2 further confirmed CCL2 inhibition of inflammation resolution in astrocytes.

Expression and clinical significance of MIP-1 alpha in serum of asthmatic patients

Objective To investigate the levels of serum macrophage inflammatory protein-1α (MIP-1α), inflammatory factors and pulmonary function in patients with asthma, and the correlation between them. Methods Eighty patients with asthma were selected who were treated in our hospital from March 2015 to August 2016, and the data of 80 healthy adults were selected as control. The differences of serum MIP-1α, inflammatory factors, immunoglobulin and lung function were observed in two groups. The correlation between MIP-1α and inflammatory factors, lung function and immunoglobulin were analyzed. Results The levels of MIP-1α, interleukin (IL)-6, and tumor necrosis factor-α (TNF-α) in patients with asthma were significantly higher than those in control group (t=-207.04, -33.209, -55.132, P<0.01); The levels of forced vital capacity (FVC), the forced expiratory volume in one second (FEV1), FEV1/FVC, maximal mid expiratory flow (MMEF), and peak expiratory flow (PEF) in the asthma group were significantly lower than those in the control group (t=17.100, 39.154, 22.791, 25.391, 19.356, P<0.01). The IgG and IgM of asthma group were significantly lower than those of the control group (t=9.564, 7.528, P<0.01). The IgE level was significantly higher than that of the control group (t=-82.683, P<0.01). The level of MIP-1α was positively correlated with the levels of IL-6, TNF-α and IgE, and negatively correlated with FVC, FEV1, FEV1/FVC, MMEF, PEF, IgG and IgM in patients with asthma. Conclusions The level of serum MIP-1α in asthmatic patients is high, and is closely related to the inflammatory cytokines and lung function in patients with asthma. Key words: Asthma; Chemokine CCL3/ME; Cytokines/ME; Inflammation; Respiratory function tests

Leukemia Cell Infiltration Causes Defective Erythropoiesis Partially through MIP-1alpha

Leukemia often results in severe anemia, which may significantly contributes to mortality and morbidity of the patients. However, the mechanisms underlying the insufficient erythropoiesis in leukemia have been poorly understood. In this study, with our recently established non-irradiated MLL-AF9 acute myeloid leukemia (AML) murine model (Cheng H et al, Blood 2015), we observed a significant decrease in hemoglobin and red blood cells (RBCs) of Peripheral blood (PB) in the leukemic mice (n=6 per group, p=0.0122 vs p=0.0003). The absolute numbers of the erythroblasts at different stages (Pro Es, Ery.A, Ery.B, Ery.C) in bone marrow (BM) were also reduced. Consistently, by gene set enrichment analysis (GSEA) of microarray data of LKS+ cells (GSE52506 ) from leukemic mice, we found significant down-regulation of erythroid differentiation related genes such as GATA1, FOG-1, LMO2 and KLF1. These genes were more significantly inhibited in megakaryocytic-erythroid progenitors (MEPs) and Pro Es from the leukemic mice. Notably, the MEPs were the most reduced subset among all the committed hematopoietic progenitor cells (HPCs) during leukemia progression (90% decrease compared to control, p = 0.0007). MEPs were gradually accumulated in the G0 phase (from 22% to 70%, p<0.001). In contrast, erythroblasts (Pro Es, Ery.A, Ery.B) were more cycling (G1/S/G2/M) in leukemic mice and the proportions of Annexin V+ cells in erythroblasts but not in MEPs were also increased during leukemia development. Colony-forming cell (CFC) assays revealed that BM plasma of leukemia mice exerted an inhibitory effect on both BFU-Es and CFU-Es of BM mononuclear cells (BMMNCs) but not on other types of colonies (40% decrease for CFU-Es, 60% decrease for BFU-Es, p<0.001). Consistently, BM plasma of AML patients could also reduce the yield of BFU-Es and CFU-Es from CD34+ cord blood cells (n=7, p=0.006).To determine which cytokines may be responsible for the inhibitory effect, we collected serum and BM plasma from control and leukemic mice for cytokine array analysis. Among the elevated cytokines, MIP-1alpha was previously reported to be up-regulated in leukemic stem cells and its higher expression was found in the majority of patients with leukemia and a subset of patients with lymphoma and myeloma according to the Oncomine data set. We also confirmed it in a cohort of untreated AML patients (n=32). Importantly, AML patients with higher expression of MIP-1alpha showed reduced survival time (median=13.08 months) compared with the patients with lower expression (median=25.86 months) based on the leukemia-gene-atlas (LGA) analysis (n=72). By the CFC assay and single cell culture with different subsets of hematopoietic stem cells (HSCs) and HPCs, MIP-1alpha was able to largely mimic the inhibitory effect on the erythroid differentiation at both stem cell and progenitor cell levels. Mechanistically, we observed higher expression of MIP-1alpha receptor CCR1 in HSCs, MEPs and erythroblasts than CCR5. Administration of CCR1 antagonist, BX471 could partially recover the yield of erythroid colonies after treatment of MIP-1alpha or leukemia BM plasma. An increase of phosphorylation of p38 (phos p38) and resulted down-regulation of GATA1 after MIP-1alpha treatment were documented by Western blots and immunostaining. In summary, our results demonstrate that leukemic cell infiltration causes severe inhibition of erythropoiesis largely at different erythroid precursor levels and this inhibitory effect is at least partially medicated by elevated MIP-1alpha level via CCR1-p38 activation in the leukemic microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Depletion of Dendritic Cells and Lack of Expression of MIP3alpha in Oral Hairy Leukoplakia

Depletion of Dendritic Cells and Lack of Expression of MIP3alpha in Oral Hairy Leukoplakia

Bone marrow plasma macrophage inflammatory protein protein-1 alpha(MIP-1 alpha) and sclerostin in multiple myeloma: Relationship with bone disease and clinical characteristics

The aim of the study was to investigate the expression of MIP-1 alpha and sclerostin in bone marrow of patients with multiple myeloma (MM), the possible association of the sclerostin and MIP-1 alpha with MBD and the clinical characteristics. 53 patients (29 M, 24 F), median age 64 years was studied. MIP-1 alpha, sclerostin and bone-specific alkaline phosphatase (bALP) levels were quantified using an enzyme-linked immunosorbent assay (ELISA). Sclerostin and MIP-1 alpha mRNA expression was determined by RT-PCR. PTH and 1,25(OH) 2D3 levels were measured with an electrochemiluminescence immunoassay. The sclerostin and MIP-1 alpha concentrations in patients with MM were higher than those in the controls. RT-PCR analysis verified that the bone marrow mononuclear cells (BMMNCs) of most patients showed sclerostin and MIP-1 alpha mRNA expression. The sclerostin and MIP-1 alpha levels in patients with ISS stage III disease were significantly higher than those in patients with ISS stage II disease (p=0.01 and 0.06). The sclerostin and MIP-1 alpha levels in patients with BMD in group C were significantly higher than those in group A+B. There was positive correlation between sclerostin levels and MIP-1 alpha, beta2-microglobulin and aCa levels. A negative association was seen between sclerostin levels and bALP, HB and ALB levels. The MM patients with high sclerostin levels (>0.72 ng/ml) had significantly shorter median survival than those with low sclerostin levels (≤0.72 ng/ml) (χ(2)=7.574, p=0.006). Our findings support the positive relationship between sclerostin levels and MIP-1alpha levels deserve further detailed research.

Tuberculosis-specific CD4+ and CD8+ T cells maintained in older latently infected individuals (43.27)

Abstract One risk factor for reactivation of latent M. tuberculosis (MTB) is old age. Although there are significant data in elderly and nursing home patients on clinical matters such as use of two-step PPD testing, there are a severe paucity of data on changes in MTB-specific immune responses that occur with aging. We have studied MTB-specific immune responses using 13-color intracytoplasmic flow cytometry analysis of CD4+ and CD8+ memory T cell subsets simultaneously examining their expression of IFN-gamma, IL-2, MIP1-alpha, and perforin after stimulation with the MTB-antigen PPD. Data from 13 latently MTB-infected PPD+ older subjects (mean age 77 range 61-89) showed that MTB-specific responses from CD4+ and CD8+ T cells were maintained compared to 8 younger PPD+ subjects (mean age 31 range 23-40). Surprisingly, frequencies of MTB-specific IL-2 secreting effector and central memory CD4+ T cell were higher in older subjects (mean 1.1%) than in younger persons (mean 0.22%, p&amp;lt;0.05). In older subjects, many of these MTB-specific CD4+ T cells only secreted IL-2 after stimulation while there was no defect in their ability to produce the full range of cytokines studied when stimulated with mitogen. Overall, as there was no defect in MTB-specific T cells in older persons, these data support a need to further broaden the T cell studies as well to expand to other areas such as potential deficits in macrophage function.

Polyfunctional capacity of T cell function in older adults (47.10)

Abstract The precise polyfunctional capabilities of T cells from older persons are not well known. We determined the polyfunctionality of memory CD8+ and CD4+ T cell subsets using a 13-color flow cytometric assay that simultaneously analyzes IFN-gamma, TNF, MIP1-alpha, IL-2, and perforin expression after stimulation. PBMC from 37 older (mean age 82) and 30 younger (mean age 34) individuals were stimulated with a sub-maximal concentration of the mitogen SEB. Older adults were also assessed by the Fried frailty test. The proportion of highly polyfunctional cells that could perform 3, 4, or 5 functions was similar in the memory T cell subsets from older and younger individuals. The percentage of cells capable of performing each individual function had only small differences between the age groups. Within the older group however, polyfunctionality of central memory CD4+ and CD8+ T cells and CD8+ effector cells had in inverse correlation with frailty while not having a relationship with age. In conclusion, as a whole the frequency and polyfunctionality and mitogen-activated CD4+ and CD8+ memory T cell subsets in older individuals is preserved compared to the younger group. Therefore, the phenomenon of immunosenescence particularly in the non-frail elderly may not include the loss of capability and breadth of function of T cells if stimulated sufficiently. This may inform specific strategies to boost the older immune system.

Dietary arginine and repeated handling increase disease resistance and modulate innate immune mechanisms of Senegalese sole ( Solea senegalensis Kaup, 1858)

Stress is known to impair immune function and disease resistance in fish. In the present study, repeated handling was employed as a chronic stressor in order to verify whether its attributed immunosuppressive effects could be minimized by dietary arginine supplementation. Therefore, Senegalese sole ( Solea senegalensis) were air exposed daily for 3 min during 14 days (handling) or left undisturbed (control). In addition, both control and handled specimens were fed 3 diets with graded levels of arginine (Arg 4.4, Arg 5.7 and Arg 6.9 g 16 g −1 N). Following the 14 days stress challenge and feeding on those diets, fish were infected with Photobacterium damselae subsp. piscicida (strain PC566.1; LD 50 5 × 10 3 cfu mL −1) and fed the same experimental diets. Respiratory burst activity and nitric oxide production of head–kidney leucocytes increased parallel to dietary arginine supplementation. HIF-1, HAMP-1, MIP1-alpha and gLYS expression values and some humoral parameters augmented in control specimens fed the Arg 5.7 and Arg 6.9 diets. Interestingly, repeated acute stress increased both disease resistance and some innate immune mechanisms in handled fish. The role of dietary arginine and repeated handling on Senegalese sole innate immunity and disease resistance are discussed.

Whole Blood Transcriptomics in Cardiac Surgery Identifies a Gene Regulatory Network Connecting Ischemia Reperfusion with Systemic Inflammation

BackgroundCardiac surgery with cardiopulmonary bypass (CS/CPB) is associated with increased risk for postoperative complications causing substantial morbidity and mortality. To identify the molecular mechanisms underlying CS/CPB-induced pathophysiology we employed an integrative systems biology approach using the whole blood transcriptome as the sentinel organ.Methodology/Principal FindingsTotal RNA was isolated and globin mRNA depleted from whole blood samples prospectively collected from 10 patients at time points prior (0), 2 and 24 hours following CS/CPB. Genome-wide transcriptional analysis revealed differential expression of 610 genes after CS/CPB (p<0.01). Among the 375 CS/CPB-upregulated genes, we found a gene-regulatory network consisting of 50 genes, reminiscent of activation of a coordinated genetic program triggered by CS/CPB. Intriguingly, the highly connected hub nodes of the identified network included key sensors of ischemia-reperfusion (HIF-1alpha and C/EBPbeta). Activation of this network initiated a concerted inflammatory response via upregulation of TLR-4/5, IL1R2/IL1RAP, IL6, IL18/IL18R1/IL18RAP, MMP9, HGF/HGFR, CalgranulinA/B, and coagulation factors F5/F12 among others. Differential regulation of 13 candidate genes including novel, not hitherto CS/CBP-associated genes, such as PTX3, PGK1 and Resistin, was confirmed using real-time quantitative RT-PCR. In support of the mRNA data, differential expression of MMP9, MIP1alpha and MIP1beta plasma proteins was further confirmed in 34 additional patients.ConclusionsAnalysis of blood transcriptome uncovered critical signaling pathways governing the CS/CPB-induced pathophysiology. The molecular signaling underlying ischemia reperfusion and inflammatory response is highly intertwined and includes pro-inflammatory as well as cardioprotective elements. The herein identified candidate genes and pathways may provide promising prognostic biomarker and therapeutic targets.

Type 1 T-cell responses in chlamydial lung infections are associated with local MIP-1α response

Chemokines and their receptors are important mediators of leukocyte trafficking and recruitment and sometimes work as modulators of T-cell responses during infections and inflammation. Modulating the biological activity of chemokines has been found to influence the course of diseases. However, little is known about the role of chemokine responses during chlamydial lung infections. We therefore analyzed the dynamics of multiple chemokines, which are frequently associated with type 1 (Th1) T cell immune responses, and their receptors for their expression in the lungs during Chlamydia muridarum (Cm) infections. We also examined the relationship between chemokine responses and the development of Th1 responses as well as the clearance of infection. Our results showed that in parallel with the high levels of gamma interferon (IFN-gamma) and IL-12 production in the lungs and draining lymph nodes, and the expansion of IFN-gamma-producing CD4 and CD8+ T cells, the production of the cell-related chemokines RANTES, IFN-gamma-inducible protein-10 (IP-10) and macrophage inflammatory protein-1alpha (MIP-1alpha) and their receptor CCR1 was elevated in the lung tissues after infection. Interestingly, in a later phase of infection, the expression of RANTES and IP-10 remained elevated but the expression of MIP-1alpha and CCR1 decreased to a low level, which suggests a closer association with the pattern of Th1 cytokine responses in the process of infection. These results suggest a close association between the MIP-1alpha response and the Th1-type T-cell responses in chlamydial lung infections.

Restoration of dysregulated CC chemokine signaling for monocyte/macrophage chemotaxis in head and neck squamous cell carcinoma patients by neem leaf glycoprotein maximizes tumor cell cytotoxicity

Previous studies have shown that the CC chemokine receptor CCR5 is downregulated on monocyte/macrophage (MO/Mphi) surfaces in head and neck squamous cell carcinoma (HNSCC) patients (stage IIIB). Ligands (RANTES, MIP-1alpha and MIP-1beta) of this chemokine receptor were also secreted in lesser quantity from MO/Mphi of HNSCC patients in comparison with healthy individuals. In an aim to restore this dysregulated receptor-ligand signaling, we have used neem leaf glycoprotein (NLGP), a novel immunomodulator reported from our laboratory. NLGP upregulated CCR5 expression, as evidenced from studies on MO/Mphi of peripheral blood from HNSCC patients as well as healthy individuals. Expression of RANTES, MIP-1alpha and MIP-1beta was also upregulated following NLGP treatment of these cells in vitro. Interestingly, NLGP has little effect on the expression of CCR5 and the ligand RANTES in oral cancer cells. This restored CCR5 receptor-ligand signaling seen in MO/Mphi was reflected in improved CCR5-dependent, p38 mitogen-activated protein kinase (MAPK)-mediated migration of MO/Mphi after NLGP treatment to a standard chemoattractant. NLGP also induces better antigen presentation and simultaneous costimulation to effector T cells by MO/Mphi by upregulating human leucocyte antigen (HLA)-ABC, CD80 and CD86. In addition, NLGP-treated MO/Mphi-primed T cells can effectively lyse tumor cells in vitro. The effects of NLGP on monocyte migration and T cell-mediated oral tumor cell killing were further demonstrated in transwell assays with or without CCR5 neutralization. These results suggest a new approach in cancer immunotherapy by modulating dysregulated CCR5 signals from MO/Mphi.

Expression of MIP-1alpha (CCL3) by a recombinant rabies virus enhances its immunogenicity by inducing innate immunity and recruiting dendritic cells and B cells.

Previously, we showed that overexpression of MIP-1alpha in mouse brain further decreased rabies virus (RABV) pathogenicity (L. Zhao, H. Toriumi, Y. Kuang, H. Chen, and Z. F. Fu, J. Virol., 83:11808-11818, 2009). In the present study, the immunogenicity of recombinant RABV expressing MIP-1alpha (rHEP-MIP1alpha) was determined. It was found that intramuscular immunization of BALB/c mice with rHEP-MIP1alpha resulted in a higher level of expression of MIP-1alpha at the site of inoculation, increased recruitment of dendritic cells (DCs) and mature B cells into the draining lymph nodes and the peripheral blood, and higher virus-neutralizing antibody titers than immunization with the parent rHEP and recombinant RABVs expressing RANTES (CCL5) or IP-10 (CXCL10). Our data thus demonstrate that expression of MIP-1alpha not only reduces viral pathogenicity but also enhances immunogenicity by recruiting DCs and B cells to the site of immunization, the lymph nodes, and the blood.

Absence of macrophage inflammatory protein-1{alpha} does not impact macrophage accumulation in adipose tissue of diet-induced obese mice.

Macrophages and T-lymphocytes are known to accumulate in the white adipose tissue (WAT) of obese mice and humans, but the factors that cause this infiltration are not yet determined. Chemokines, which attract leukocytes to inflammatory sites, are candidates for this process. Macrophage inflammatory protein-1alpha (MIP-1alpha) expression is significantly elevated in WAT of obese mice and humans and positively correlates with fasting plasma insulin, but its potential role in leukocyte recruitment to WAT is unknown. MIP-1alpha-deficient, heterozygous, and wild-type mice were fed a Western diet (WD) for 16 wk. Plasma lipids, adipose tissue mass, energy expenditure, food intake, liver triglyceride content, and inflammatory cytokine expression were not different among genotypes. Gene expression of macrophage markers F4/80 and CD68, as well as T-lymphocyte marker CD3epsilon was increased in perigonadal WAT of obese WD-fed mice but was not influenced by MIP-1alpha expression level. Immunohistochemical analysis of WAT also showed no effect of MIP-1alpha on macrophage content. Two related chemokines, MIP-1beta and RANTES, had reduced expression in obese male MIP-1alpha-deficient mice compared with wild-type controls (P < or = 0.05). In mice fed the WD for 6 wk, WAT macrophage content was unchanged; however, CD8+ T-lymphocytes accumulated to a lesser extent in the MIP-1alpha-null mice. Therefore, expression of MIP-1alpha, as well as that of MIP-1beta and RANTES, increases as a consequence of weight gain, but these chemokines may not be required for the recruitment of monocytes to WAT during diet-induced obesity in mice and may impact T-lymphocyte recruitment only at early time points after WD feeding.

18β-Glycyrrhetinic Acid Ameliorates Acute Propionibacterium acnes-induced Liver Injury through Inhibition of Macrophage Inflammatory Protein-1α

18Beta-glycyrrhetinic acid (GA), the major bioactive component of licorice root extract, has a protective effect on hepatic injury and exhibits antiinflammatory activity. Here, we investigate the effect of GA in Propionibacterium acnes-induced acute inflammatory liver injury. C57BL/6 mice were primed with P. acnes followed by lipopolysaccharide challenge to induce fulminant hepatitis. GA (75 mg/kg) or vehicle control was administered intraperitoneally daily 1 day after P. acnes priming, and GA significantly improved mouse mortality. Then, to investigate the underlying mechanisms of GA in this acute inflammatory liver injury model, we primed C57BL/6 mice with P. acnes only. We propose that GA ameliorates acute P. acnes-induced liver injury through reduced macrophage inflammatory protein (MIP)-1alpha expression in Kupffer cells by down-regulating MyD88 expression and inhibiting NF-kappaB activation. Reduced MIP-1alpha expression lowered the recruitment of CD11c(+)B220(-) dendritic cell precursors into the liver. Consequently, GA treatment inhibits the activation and proliferation of liver-infiltrating CD4(+) T cells and reduces the production of serum alanine aminotransferase and proinflammatory cytokines such as interferon-gamma and tumor necrosis factor-alpha. Moreover, anti-MIP-1alpha treatment in P. acnes-primed mice inhibits the recruitment of dendritic cell precursors into the liver and suppresses mouse mortality as GA does. Taken together, our results suggest that GA exhibits antiinflammatory effects through inhibition of MIP-1alpha in a mouse model of acute P. acnes-induced inflammatory liver injury.

Varicose Veins Show Enhanced Chemokine Expression

Leucocyte infiltration in the wall of varicose veins has been reported previously. This study was designed to investigate the expression of pro-inflammatory cytokines and chemokines in control and in patients with varicose veins and to test the effect of treating varicose vein patients with acetylsalicylic acid (ASA) on cytokine expression prior to removal of varices. Sections of vein were removed during operation from both patient groups, and ribonuclease protection assays (RPAs) were performed to assess the expression of chemokines. Group I included non-varicose saphenous veins from healthy patients undergoing amputation for trauma. Varicose veins were obtained from patients with primary varicose undergoing surgical treatment who received no drug (group II) or treatment with 300 mg day(-1) of ASA for 15 days before surgery (group III). Non-varicose veins constitutively expressed low levels of monocyte-chemoattractant protein (MCP-1) and interleukin (IL)-8 mRNA. Varicose veins had a distinct chemokine expression pattern, since significant up-regulation of MCP-1 and IL-8 and a marked expression of IP-10, RANTES, MIP-1alpha and MIP-1beta mRNA were detected. Removal of the endothelium did not alter this pattern. Varicose veins obtained from patients treated with ASA showed a consistent decrease in chemokine expression, although it did not reach statistical significance. Varicose veins showed increased expression of several chemokines compared to control veins. A non-significant reduction of activation was observed following treatment with ASA for 15 days.

CCR3, CCR2A and macrophage inflammatory protein (MIP)‐1α, monocyte chemotactic protein‐1 (MCP‐1) in the mouse hippocampus during and after pilocarpine‐induced status epilepticus (PISE)

To investigate protein and gene expressions of chemokine subtypes CCR3, CCR2A and their respective ligands macrophage inflammatory protein 1-alpha (MIP-1alpha), monocyte chemotactic protein-1 (MCP-1) in the normal mouse central nervous system (CNS) and in the hippocampus at different time points during and after pilocarpine-induced status epilepticus (PISE). CCR3 and MIP-1alpha protein expressions were mapped in the mouse CNS. The protein and gene expressions of CCR3 and CCR2A and their respective ligands MIP-1alpha, MCP-1 in the hippocampus were studies by immunocytochemical and quantitative real-time RT-PCR during and after PISE. CCR3 and MIP-1alpha gene expression and immunopositive neurones were broadly distributed in the CNS. CCR3 and CCA2A gene and their protein expression were downregulated in the hippocampus at 1 h during PISE. The protein expression of MIP-1alpha, MCP-1 decreased but gene expression increased at 2 h during PISE. In the hilus of the dentate gyrus, significant reduction of the numbers of CCR3, CCR2A, MCP-1 immunopositive neurones occurred from 1 h during to 2 months after PISE, but the number of MIP-1alpha neurones reduced from 2 h during to 2 months after PISE. Induced expression of CCR3 at 1 week, CCR2A, MCP-1 or MIP-1alpha at 1 week and 2 months after PISE was found in reactive astrocytes. MCP-1 was also demonstrated in the blood vessels of the hippocampus at 2 months after PISE. CCR3 and MIP-1alpha may play important functional roles in the mouse brain. The downregulation of CCR3, CCR2A, MIP-1alpha and MCP-1 in the hippocampal neurones at the acute stage during and after PISE may weaken the neuroprotective mechanisms. However, induced expression of MCP-1 in hippocampal blood vessel may be related to changes in permeability of the blood-brain barrier during epileptogenesis.

WC1+γδ T Cells Indirectly Regulate Chemokine Production DuringMycobacterium bovisInfection in SCID-bo Mice

Previous studies have suggested an important role for WC1 (+)gammadelta T cells in the regulation of mycobacterial-induced inflammation in the spleen and liver of heterochimeric SCID-bovine (SCID-bo) mice. To examine the role of these cells, we investigated the levels of selected chemokines and IL12-p70 post-infection in reconstituted SCID-bo mice. Mice were treated with a monoclonal antibody specific for boWC1 to eliminate WC1-bearing cells. Isotype control treated or bovine gammadelta TCR-depleted mice were assayed in parallel. Following infection with Mycobacterium bovis, mice were examined post-infection for the expression of IL12-p70, IP-10, MIP-1alpha, lymphotactin and MIG by ELISA in plasma and from activated splenocytes. Treatment with the anti-bovine WC1 resulted in reduced serum plasma levels of IP-10, MCP-1, and IL-12p70 versus control mice. The potential of WC1 (+)gammadelta TCR-bearing cells to produce chemokines and cytokines was determined directly from peripheral blood of cattle. Our results indicate that these cells have a fairly restricted capability to produce the chemokines examined in SCID-bo mice, but may be a significant source of cytokines (IL-2, IL-10, IL-12, IL-15, and IFNgamma) and contribute to cytotoxicity through expression of FasL and perforin. In M.bovis-infected liver tissue, depletion of the WC1(+) subset was associated with increased numbers of CD3(+)T cells adjacent to venules and portal tracts. These results suggest that the WC1(+) subset in cattle may contribute to chemotaxis through indirect effects on chemokine levels. Further, activated WC1(+)gammadelta TCR(+) cells up-regulate cytokines with direct regulatory effects on T cell and macrophage function and express effector molecules with critical roles in cytotoxicity.

Immunoexpression of Macrophage Inflammatory Protein-1 Alpha on Bone Marrow Trephine Biopsies Correlates with the Extent of Bone Disease in Newly Diagnosed Patients with Multiple Myeloma

Macrophage inflammatory protein-1 alpha (MIP-1alpha) is a low molecular weight chemokine that belongs to the RANTES family and is a potent osteoclast stimulator. Previous studies have shown that malignant plasma cells (PCs) from myeloma cell lines produce MIP-1alpha, while MIP-1alpha levels are elevated in the bone marrow plasma and the serum of patients with multiple myeloma (MM) and correlate with the extent of bone disease. The purpose of our study was to evaluate the role of MIP-1alpha immunoexpression on bone marrow trephine biopsies in myeloma bone disease and examine possible correlations between MIP-1alpha expression with survival and prognostic factors of MM. We evaluated formalin fixed paraffin-embedded bone marrow sections of 130 patients with newly diagnosed MM (66M/64F, median age: 68 years). Bone marrow sections were subjected to immunohistochemistry study using the anti-MIP- 1alpha monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The immunoreactivity of MIP-1alpha was examined on the basis of positive PCs with the following cut off values: <20% positive PCs (negative expression group, group I), 20–50% positive PCs (intermediate expression group, group II) and >50% positive PCs (high expression group, group III). Moreover MIP-1alpha was measured using ELISA methodology (R&D Systems, Minneapolis, MN, USA) in the serum of the patients. The extent of bone disease was assessed radiographically, and grading was performed as follows: group A included 43 patients (33%) who had no lytic lesions and/or osteoporosis only, group B included 24 patients (18%) who had lytic lesions in 1–3 areas, whereas group C included 63 patients (48%) with lytic lesions in >3 areas and/or a bone fracture. Thirty-seven (28%) patients had negative MIP-1alpha expression, 17 (13%) intermediate expression and 79 (59%) high expression of MIP-1alpha. MIP-1alpha expression of ≥20% PCs in the trephine biopsies significantly correlated with the extent of bone disease (Figure 1, ANOVA p<0.0001). Furthermore, 91 patients (70%) had values of serum MIP-1alpha >14 pg/ml, which was the median value observed in 20 controls of similar age and gender (p<0.001). MIP-1alpha serum levels also correlated with the extent of bone disease (ANOVA p<0.0001). In terms of overall survival, no significant association was observed in relation to MIP-1alpha expression. Increased immunoexpression of MIP-1alpha was associated with high plasma cell infiltration in the bone marrow (r=0.348, p<0.0001), low platelet count (r=0.282; p=0.0012), hypercalcemia (r=0.246; p=0.022), elevated serum creatinine (r=0.258, p=0.027), and advanced disease stage (ISS, p=0.034). Our findings underline, for the first time, the increased incidence of high immunoexpression of MIP-1alpha in the malignant PCs of myeloma patients and the correlation between the expression of MIP-1alpha by myeloma PCs in the trephine biopsies and the extent of lytic bone disease. These results confirm the significant role of MIP-1alpha in the pathogenesis of myeloma bone disease and suggest that MIP-1alpha may represent a rational therapeutic target for the management of bone destruction in myeloma. [Display omitted]

Strong Correlation Between Angiogenesis and Macrophage Counts in Waldenstrom's Macroglobulinemia: Implications into the Biology of the Disease

Angiogenesis represents an essential step of disease progression in several hematological malignancies. A Mayo Clinic study reported that microvessel density (MVD) was increased (intermediate- or high- grade angiogenesis) in 30% of patients with Waldenstrom s Macroglobulinemia (WM), showed only weak correlation with marrow infiltration and had no impact on patients' survival [Rajkumar et al, Semin Oncol 2003; 30:262-4]. Macrophage inflammatory protein-1 alpha (MIP-1alpha) is a potent chemoattractant for macrophages, which contributes to increased angiogenesis in malignant diseases, including multiple myeloma. Our group has reported that serum levels of MIP-1alpha are elevated in WM. To further elucidate the role of angiogenesis in WM, we investigated the association between MVD, MIP-1alpha expression and the macrophage numbers in trephine biopsies of 34 patients with newly-diagnosed WM (3 with asymptomatic disease) and 3 with IgM-Monoclonal Gammopathy of Undetermined Significance (MGUS). Bone marrow biopsies were studied using double immunohistochemical staining for CD34 (endothelial cells) and CD68 (macrophages/mast cells) using antibodies from Becton Dickinson, San Jose, CA, USA & Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, respectively. We have also used double immunohistochemical staining for CD20/MIP-1alpha and for CD138/MIP-1alpha using an anti-MIP-1alpha antibody from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) to evaluate the MIP-1alpha expression by WM cells. Thirteen patients (35%) showed intermediate-grade and 4 (10%) high-grade angiogenesis. All patients with IgM-MGUS and asymptomatic WM had a very low microvessel count (median: 1, range: 1–2), while the median microvessel count for symptomatic WM was 4 (range: 1–8, p<0.01). There was a strong correlation between the grade of angiogenesis (as assessed by the microvessel counts) and the number of macrophages into the “hot-spots” (r=0.823, p<0.0001). Furthermore, statistically significant correlations were observed between the percentage of lymphoplasmacytoid cell infiltration of the bone marrow with microvessel counts (r=0.554, p=0.002) and macrophage numbers into the “hot spots” (r=0.457, p=0.011). WM patients with intermediate or high grade angiogenesis had increased IgM levels (p=0.007), lower hemoglobin levels (p=0.024), and reduced platelet counts (p=0.043), while patients with high-grade angiogenesis had a tendency to higher incidence of lymphadenopathy compared with all others (3/4, 75% vs. 9/33, 27%; p=0.054). There was no correlation between angiogenesis and survival. We have also observed that WM cells of all patients produced MIP-1alpha. CD138 positive WM cells had higher expression of MIP-1alpha compared to CD20 positive WM cells (p=0.001). Patients with increased numbers of CD68 positive macrophages had increased expression of MIP-1alpha by their WM cells (r=0.732, p<0.001). The results of our on going study suggest that WM cells produce MIP-1alpha to attract macrophages in their bone marrow microenvironment. These macrophages seem to play a significant role in the angiogenesis process and are possibly implicated into the biology of WM.

MIP-1&amp;#945; and MCP-1 Induce Migration of Human Umbilical Cord Blood Cells in Models of Stroke

Monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein (MIP-1alpha) are implicated in monocyte infiltration into the central nervous system (CNS) under pathological conditions. We previously showed that in vivo human umbilical cord blood cells (HUCB) migrate toward brain injury after middle cerebral artery occlusion (MCAO). We hypothesized that MCP-1 and MIP-1alpha may participate in the recruitment of HUCB towards the injury. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO), and 24 hours later the production of MCP-1 and MIP-1alpha in the brain was examined with immunohistochemistry, ELISA, and western blotting. The chemotactic effect of MCP-1 and MIP-1alpha, and the expression of MCP-1 receptor CCR2 and MIP-1alpha receptor CCR1, CCR5 on the surface of HUCB were also examined. MCP-1 and MIP-1alpha expression were significantly increased in the ischemic hemisphere of brain, and significantly promoted HUCB cell migration compared to the contralateral side. This cell migration was neutralized with polyclonal antibodies against MCP-1 or MIP-1alpha. Also chemokine receptors were constitutively expressed on the surface of HUCB cells. The data suggested that the increased chemokines in the ischemic area can bind cell surface receptors on HUCB, and induce cell infiltration of systemically delivered HUCB cells into the CNS in vivo.