An important role for the chemokine macrophage inflammatory protein-1 alpha in the pathogenesis of the T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated, inflammatory demyelinating disease of the central nervous system (CNS) that serves as a model for the human demyelinating disease, multiple sclerosis. A critical event in the pathogenesis of EAE is the entry of both Ag-specific T lymphocytes and Ag-nonspecific mononuclear cells into the CNS. In the present report we investigated the role of two C-C chemokines (macrophage inflammatory protein-1 alpha (MIP-1 alpha) and monocyte chemotactic protein-1) and a C-x-C chemokine (MIP-2) in the pathogenesis of EAE. Production in the CNS of MIP-1 alpha, but not that of MIP-2, a rodent homologue of IL-8, or monocyte chemotactic protein-1, correlated with development of severe clinical disease. Administration of anti-MIP-1 alpha, but not that of anti-monocyte chemotactic protein-1, prevented the development of both acute and relapsing paralytic disease as well as infiltration of mononuclear cells into the CNS initiated by the transfer of neuroantigen peptide-activated T cells. Ab therapy could also be used to ameliorate the severity of ongoing clinical disease. Anti-MIP-1 alpha did not affect the activation of encepahlitogenic T cells as measured by cytokine secretion, surface marker expression, and ability to adoptively transfer EAE. These results demonstrate that MIP-1 alpha plays an important role in directing the chemoattraction of mononuclear inflammatory cells in the T cell-mediated autoimmune disease, EAE.     

The production of macrophage inflammatory protein-1 alpha by human basophils

Macrophage inflammatory protein-1alpha (MIP-1alpha) has previously been shown to be produced by mononuclear cells, eosinophils, and neutrophils. Its production by basophils has not been investigated. The objective of this study was to investigate the production of MIP-1alpha by basophils. Peripheral blood basophils were separated by Percoll gradient centrifugation, cultured overnight, and processed for double immunocytochemistry using Abs against MIP-1alpha and FcepsilonRIalpha (alpha subunit of IgE receptor type 1). We demonstrated that basophils expressed immunoreactive MIP-1alpha upon stimulation with anti-IgE. Less than 5% of the basophils stained for MIP-1alpha without stimulation. The secretion of MIP-1alpha by basophils was studied by ELISA. In these experiments, basophils were further enriched to 65 to 99% (median, 86%) by a negative selection method. Basophils released MIP-1alpha when stimulated by Abs against IgE and FCepsilonRIalpha as well as IL-3 and the calcium ionophore, A23187. In parallel experiments, PBMC, eosinophils, and neutrophils did not produce MIP-1alpha in response to anti-IgE, but they did so in response to A23187. No MIP-1alpha release was detected in platelet preparations. Preincubation with IL-3 (15 min or 18 h) augmented anti-IgE-included basophil MIP-1alpha production. The secretion of MIP-1alpha by basophils was detectable shortly after stimulation and gradually increased over 24 h. Since MIP-1alpha has potent inflammatory and histamine-releasing activities, its production by basophils may indicate a positive feedback mechanism for allergic inflammation.     

Expression of chemokines RANTES, MIP-1alpha and GRO-alpha correlates with inflammation in acute experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an investigator-initiated disorder that serves as an animal model for the common human demyelinating disease multiple sclerosis. Both diseases are typified by disseminated perivascular and submeningeal cuffs in the central nervous system (CNS). It was shown recently that chemokines are integral to the pathogenesis of EAE. In the present study we analyzed the gene expression of three chemokines, RANTES, MIP-1alpha and GRO-alpha, at the onset of acute EAE, and correlated that expression with the intensity of inflammatory changes in the CNS. We showed that all three chemokines are upregulated simultaneously with symptom onset of acute EAE, and that chemokine expression correlates with the intensity of inflammation in the CNS. This consistent relationship supports the hypothesis that chemokines are relevant to leukocyte accumulation in CNS parenchyma.     

Characterisation of macrophage inflammatory protein-5/human CC cytokine-2, a member of the macrophage-inflammatory-protein family of chemokines

The aim of this study was to determine the level of endogenous prostaglandin E2 (PGE2), prostaglandin F1 alpha (6-keto-PGF1 alpha) and thromboxane B2 (TXB2) in the gastric and duodenal mucosa of patients with duodenal ulcer and duodenitis. Besides, the investigation aimed at determining the effect of smoking and infection by Helicobater pylori on prostaglandin synthesis. The investigation comprised 62 patients with duodenal ulcer, 46 patients with duodenitis and 44 controls. The results of our investigation indicate that the decreased prostaglandin synthesis in gastric and duodenal mucosa determined in patients with duodenal ulcer may have a considerable role in development of duodenal ulcer. Furthermore, the harmful effects of smoking on the gastric and duodenal mucosa may be mediated by the decreased prostaglandin synthesis in the gastric and duodenal mucosa. However, Helicobacter pylori seems to affect the development of duodenal ulcer through other mechanisms.     

Sequential dynamics of monocyte chemotactic protein-1 expression in herniated nucleus pulposus resorption

We previously demonstrated that the granulation tissues of herniated nucleus pulposus are composed of a marked infiltration of macrophages that strongly express monocyte chemotactic protein-1. Monocyte chemotactic protein-1 is a chemotactic cytokine that contributes to the activation and recruitment of macrophages. Relatively little is known about its role in the resorption process of herniated nucleus pulposus. To clarify the sequential dynamics of expression of monocyte chemotactic protein-1 in the granulation tissues of herniated nucleus pulposus, we introduced a rat autologous transplantation model of nuclear materials onto its lumbar dura mater and performed immunohistological analysis and competitive polymerase chain reaction assay using the grafted samples. Immunohistological analysis demonstrated that the majority of infiltrating mononuclear cells expressed monocyte chemotactic protein-1. Monocyte chemotactic protein-1 mRNA was expressed in the first 3 weeks after the procedure and was significantly and maximally upregulated at 1 week. To determine whether human recombinant monocyte chemotactic protein-1 facilitates the resorption process of herniated nucleus pulposus, we introduced another model of autologous transplantation, wherein the nuclear materials were grafted to the abdominal subcutaneous tissues and recombinant monocyte chemotactic protein-1 was subsequently applied to these materials. When monocyte chemotactic protein-1 was injected into the murine nucleus pulposus tissues, they reduced in size more rapidly than in the control group. These findings suggest that monocyte chemotactic protein-1 plays an important role in the recruitment of macrophages in the early phase of the resorption process of herniated nucleus pulposus and that its application may physiologically facilitate the resorption process of the nucleus pulposus.     

Cytokine-induced production of macrophage inflammatory protein-1alpha (MIP-1alpha) in cultured human astrocytes

The human cytotoxic T cell line TALL-104 displays potent anti-tumor effects in animals with spontaneous and induced malignancies. We investigated the biodistribution of TALL-104 cells in tumor bearing and healthy mice. 111In-labeled TALL-104 cells, injected intravenously, localized primarily in the lungs for the first 2 h, and redistributed to liver, spleen, and kidneys in the following 24 h both in immunodeficient and immunocompetent mice. TALL-104 cells consistently accumulated in the tumor and at sites of metastases. In animals injected with free 111In-oxine, most of the radioactivity remained in the blood pool with no significant organ accumulation. These data support the tumor homing properties of TALL-104 cells, information which is crucial to their therapeutic efficacy in forthcoming clinical trials.     

Alternative macrophage activation-associated CC-chemokine-1, a novel structural homologue of macrophage inflammatory protein-1 alpha with a Th2-associated expression pattern

We have cloned a novel human CC-chemokine, alternative macrophage activation-associated CC-chemokine (AMAC)-1. The isolated cDNA clone (803 bp) shows a single open reading frame of 267-bp coding for 89 amino acid residues; mature AMAC-1 protein is predicted to consist of 69 amino acids with a m.w. of 7855. Sequence alignment and 3D-modeling show the typical structural characteristics of CC-chemokines with special features in the receptor-activating domain. AMAC-1 is most closely related to MIP-1 alpha with a cDNA and protein sequence homology of 55% and 59%, respectively. However, the expression pattern of AMAC-1 is directly opposite to that of MIP-1 alpha. While MIP-1 alpha is induced by classical macrophage mediators such as LPS and is inhibited by IL-4 and glucocorticoids, AMAC-1 is specifically induced in macrophages by alternative macrophage mediators such as IL-4, IL-13, and IL-10. Expression of AMAC-1 is inhibited by IFN-gamma while glucocorticoids exert a slightly positive synergistic effect in combination with IL-4. Peripheral blood monocytes do not express AMAC-1; time course experiments show that monocyte-to-macrophage differentiation is a prerequisite for AMAC-1 expression. Expression of AMAC-1 by granulocyte-macrophage CSF/IL-4-induced, monocyte-derived dendritic cells is complex; in mature adherent dendritic cells, however, only minor AMAC-1 mRNA expression was found. In vivo, AMAC-1 is expressed by alveolar macrophages from healthy persons, smokers, and asthmatic patients. In conclusion, AMAC-1 is a novel CC-chemokine whose expression is induced in alternatively activated macrophages by Th2-associated cytokines; thus, AMAC-1 may be involved in the APC-dependent T cell development in inflammatory and immune reactions.     

Alleviation of monocrotaline-induced pulmonary hypertension by antibodies to monocyte chemotactic and activating factor/monocyte chemoattractant protein-1

Administration of monocrotaline (MCT) causes pulmonary vascular lesions consisting of monocyte/macrophage infiltration in the early phase and medial thickening in pulmonary arteries and arterioles associated with pulmonary hypertension (PH) in the later phase. However, the molecular mechanism of monocyte/macrophage infiltration and its roles remain elusive. Herein, we have evaluated the role of a potent monocyte chemotactic and activating chemokine/monocyte chemoattractant protein-1 (MCAF/MCP-1) in MCT-induced PH in rats. A single injection of MCT induced PH at Day 21, as evidenced by increases in the ratio of right ventricular to left ventricular and septum weights (RV/LV+S) and right ventricular systolic pressure (RVSP). A significant increase in macrophage number in lungs started at Day 14, reaching a maximum at Day 21. MCAF/MCP-1 levels in bronchoalveolar lavage fluids were elevated significantly at Day 14 and remained high until Day 28, whereas plasma MCAF/MCP-1 levels increased at Day 7, returning to normal levels by Day 21. Immunoreactive MCAF/MCP-1 proteins were mainly detected in macrophages in alveoli and in perivascular regions of pulmonary arterioles and venules. Intravenous administration of anti-MCAF/MCP-1 antibodies with MCT significantly decreased macrophage infiltration and eventually reduced the increases in RV/LV+S and RVSP, as well as medial thickening of pulmonary arterioles. Thus, MCAF/MCP-1 is essentially involved in MCT-induced PH by recruiting and activating macrophages.     

Expression and hormonal regulation of monocyte chemotactic protein-1 in myometrium and leiomyomata

OBJECTIVE: The pathogenesis of leiomyoma likely involves interactions of sex steroids with paracrine growth factors or cytokines resulting in modulation of local immunity. Monocyte chemotactic protein-1 (MCP-1) is a chemotactic and activating factor for monocytes and is produced by multiple tumors and has antitumor effects. We investigated the expression of MCP-I in leiomyoma and myometrium as well as the regulatory role of steroid hormones and cytokines on MCP-1 expression and the effect of MCP-1 on the proliferation of leiomyoma cells. DESIGN: Prospective study. SETTING: University medical center. PATIENT(S): Women with (n = 20) or without (n = 11) leiomyoma. INTERVENTION(S): First. MCP-1 messenger RNA (mRNA) levels in myometrium and leiomyoma were measured, and then myometrial and leiomyoma cells in culture were treated with steroid hormones and cytokines. MAIN OUTCOME MEASURE(S): The MCP-1 mRNA was evaluated by Northern analysis. Immunoreactive MCP-1 in cell cultures was quantified by ELISA. Leiomyoma cell proliferation was assessed with [3H]thymidine incorporation. RESULT(S): The MCP-1 mRNA levels in myometrial samples were 4.7-fold higher than in the leiomyoma samples. Myometrial MCP-1 mRNA levels were 2.4-fold higher in secretory than in proliferative phase samples. The highest MCP-1 levels were observed in samples from women using GnRH analogues. Estradiol and progestins, alone or in combination, resulted in a decrease in MCP-1 protein production. There was an increase in the proliferation of leiomyoma cells treated with anti-MCP-1 neutralizing antibody. CONCLUSION(S): These findings suggest that MCP-1 may have antineoplastic activity in leiomyomata and that sex steroids may be exerting their growth stimulatory effect in leiomyomata through down-regulation of MCP-1.     

Genetic analysis of inflammation, cytokine mRNA expression and disease course of relapsing experimental autoimmune encephalomyelitis in DA rats

Past history of major depression is more common in smokers than in non-smokers. We have shown in a previous study that lifetime prevalence of major depression is higher in dependent smokers and they have lower monoamine oxidase-A and -B activities than non-smokers. Because several studies have found an association between MAO-B activity and depression we analysed data of these smokers to assess whether past history of major depression is associated with reduced monoamine oxidase activities (A and B) or not. Further, we tried to characterize smokers with past history of major depression and its effect on withdrawal symptoms. The data of 88 dependent smokers (Fagerstr?m Tolerance Questionnaire score > or = 6 and smoking > or = 20 cigarettes/day) who participated in a smoking cessation study were analysed. Smokers with past history of major depression but without current illness did not differ in demographic and smoking characteristics from smokers without past history of major depression. Smokers with past history of major depression were mainly women and had lower body mass index. Adjusted for gender and body mass index dependent smokers with or without past history of depression had similar MAO-A and MAO-B activities but smokers with past history of major depression had significantly lower resting plasma norepinephrine levels. Smokers with past history of depression had not significantly higher ratings for depression (Montgomery-Asberg Depression Rating Scales) and anxiety (Hamilton Anxiety Scales) and smoking cessation did not exacerbate these ratings (assessed up to 3 months) and none had depressive episode during the postcessation period up to one year. Past history of depression was associated with higher scores on 'expressed sadness' and 'depressive mood'. Abstinent smokers with past history of depression had significantly higher ratings in one of the seven ratings of a 6 months period for craving (day 28), anxiety (day 7) and total withdrawal symptom score (day 7) when compared to those who had no past history of major depression. It is concluded that (i) past history of major depression is more frequent in female smokers; (ii) smokers with past history of depression may have more intense withdrawal symptoms (craving and anxiety) at some time after cessation: and (iii) past history of depression does not affect monoamine oxidase activities, therefore, reduced monoamine oxidase activities found in previous studies are possibly characteristic features of smoking.     

E- and P-selectin are not involved in the recruitment of inflammatory cells across the blood-brain barrier in experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE) inflammatory cells cross the endothelial blood-brain barrier (BBB) and gain access to the central nervous system (CNS). Here we show that E- and P-selectin are not involved in the recruitment of inflammatory cells across the BBB. Neither expression of E- nor P-selectin is induced in BBB-forming endothelium at any time after initiation of EAE. Some of the inflammatory cells present in the CNS during EAE express ligands for E- or P-selectin. However, anti-E- and P-selectin antibodies influence neither immigration of inflammatory cells across the BBB nor the development of EAE. In general, suppression of E- and P-selectin expression on BBB endothelium is dependent on factors derived from the CNS microenvironment, eg, astrocytes. Our results suggest that during EAE suppression of E- and P-selectin expression on the BBB provides a CNS-specific mechanism to reduce leukocyte recruitment into the CNS.     

Human retinal pigment epithelial cell interleukin-8 and monocyte chemotactic protein-1 modulation by T-lymphocyte products

PURPOSE: The purpose of the study was to examine the effect of T-lymphocyte products on human retinal pigment epithelial (HRPE) cell interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) secretion and gene expression. METHODS: HRPE cells were stimulated for 2, 4, 8, or 24 hours with 20% conditioned media (CM) from T-lymphocytes stimulated with CD3 or CD28 monoclonal antibodies (mAbs) or phorbol myristic acid. In some experiments, CM from CD3 mAb-stimulated T-lymphocytes was preincubated with neutralizing anti-(alpha)-tumor necrosis factor (TNF), alpha-interferon-gamma (IFN-gamma), or alpha-interleukin-1 (IL-1) mAb (control) to determine the contributions of each of these cytokines to HRPE chemokine induction by stimulated T-lymphocyte CM. HRPE cells were stimulated for 8 and 24 hours with IL-1 beta (0.2 to 20.0 ng/ml) (positive control), TNF-alpha (0.2 to 20.0 ng/ml) (positive control), IFN-gamma (1 to 1000 U/ml), IFN-gamma + IL-1 beta, IFN-gamma + TNF-alpha. Interleukin-2 (IL-2; 100 ng/ml) alone or in combination with IL-1 beta, TNF-alpha, or IFN-gamma also was tested. Enzyme-linked immunosorbent assay (ELISA) and Northern blot analyses were performed to determine secreted IL-8 and MCP-1 and their steady state mRNA expression, respectively. RESULTS: ELISA showed significant increases in HRPE IL-8 and MCP-1 secretion by CM from T-lymphocytes stimulated with CD3 or CD3 + CD28 mAb. Smaller, but significant, increases in IL-8 and MCP-1 resulted from CM phorbol myristic acid-stimulated T-lymphocytes. CM preincubated with neutralizing alpha-TNF or alpha-IFN-gamma mAb induced significantly less HRPE IL-8 and MCP-1, whereas preincubation of CM with neutralizing alpha-IL-1 mAb failed to inhibit CM-induced IL-8 or MCP-1. Northern blot analysis showed increased HRPE IL-8 and MCP-1 mRNA expression within 2 hours of stimulation and was maintained up to 24 hours. CM from T-lymphocytes stimulated with CD3 mAb or CD3 + CD28 mAb produced the greatest increases in IL-8 and MCP-1 mRNA. IFN-gamma induced dose-dependent increases in HRPE MCP-1, but not IL-8, IFN-gamma potentiated IL-1 beta and TNF-alpha-induced MCP-1 production, but showed little modulation of IL-1 beta and TNF-alpha-induced IL-8 production. IL-2 did not induce HRPE IL-8 or MCP-1, nor did it modulate the effects of the other cytokines. Northern blot analysis confirmed the ELISA results. CONCLUSIONS: T-lymphocyte secretions induce HRPE IL-8 and MCP-1 gene expression and secretion. TNF and IFN-gamma appear to be necessary components of T-lymphocyte CM for the induction of HRPE IL-8 and MCP-1. IFN-gamma alone induces HRPE MCP-1, albeit to a lesser extent than would IL-1 beta or TNF-alpha, and potentiates IL-1 beta- and TNF-alpha-induced HRPE MCP-1. IL-2 does not appear to modulate cytokine-induced HRPE IL-8 or MCP-1.     

Plasma levels of monocyte chemoattractant protein-1 but not those of macrophage inhibitory protein-1alpha and RANTES correlate with virus load in human immunodeficiency virus infection

Plasma levels of proinflammatory cytokines, cytokine inhibitors, and the beta chemokines RANTES, macrophage inhibitory protein (MIP)-1alpha, and monocyte chemoattractant protein (MCP)-1 were studied in relationship with virus load in 40 patients exhibiting plasma levels of HIV RNA ranging between undetectable and levels >10(6) copies/mL. Mean plasma levels of MCP-1 were increased in patients with high virus load compared with HIV-seropositive subjects with undetectable plasma viral RNA and healthy controls. MCP-1 levels were directly correlated with plasma levels of HIV RNA. No correlation was observed between virus load and plasma concentrations of MIP-1alpha and RANTES. The results suggest that low rates of viral replication in vivo are not dependent on increased production of the suppressive chemokines RANTES and MIP-1alpha. Since MCP-1 upregulates viral replication in vitro, the results may suggest a role for MCP-1 in triggering viral replication in HIV disease.     

Abl protein kinase abrogates the response of multipotent haemopoietic cells to the growth inhibitor macrophage inflammatory protein-1 alpha

OBJECTIVE: This study compared the mental health and cognitive development of 9- to 12-year-old Eritrean war orphans living in two orphanages that differed qualitatively in patterns of staff interaction and styles of child care management. METHOD: The directors and several child care workers at each institution were asked to complete staff organization and child management questionnaires. The psychological state of 40 orphans at each institution was evaluated by comparing their behavioral symptoms and performance on cognitive measures. RESULTS: Orphans who lived in a setting where the entire staff participated in decisions affecting the children, and where the children were encouraged to become self-reliant through personal interactions with staff members, showed significantly fewer behavioral symptoms of emotional distress than orphans who lived in a setting where the director made decisions, daily routines were determined by explicit rules and schedules, and interactions between staff members and the children were impersonal. CONCLUSIONS: When orphanages are the only means of survival for war orphans, a group setting where the staff shares in the responsibilities of child management, is sensitive to the individuality of the children, and establishes stable personal ties with the children serves the emotional needs and psychological development of the orphans more effectively than a group setting that attempts to create a secure environment through an authoritative style of management with explicit rules and well-defined schedules.     

Expression of E-selectin, sialyl Lewis X, and macrophage inflammatory protein-1alpha by colonic epithelial cells in ulcerative colitis

The pathogenic significance of cell adhesion molecules (CAMs) in ulcerative colitis (UC) is largely unknown. Colonic expression of E-selectin, sialyl Lewis X (sLe(x)), and macrophage inflammatory protein-1x (MIP-1alpha) as well as serum concentrations of E-selectin and MIP-1alpha in UC were studied. Thirty patients with UC, 10 patients with irritable bowel syndrome, and 10 healthy subjects were included. Colonic biopsies were stained immunohistochemically, and blood concentrations were measured with an ELISA technique. Soluble E-selectin did not correlate with diagnosis or disease activity. MIP-1alpha was below the detection limit. Epithelial cells expressed all three molecules, both on surface membranes and intracellularly. sLe(x) staining was weaker (P = 0.0002) and MIP-1alpha staining stronger (P = 0.014) in UC patients than in controls. Leukocyte MIP-alpha staining correlated with diagnosis (P = 0.021), sLe(x) staining (P = 0.023), and colonoscopy (P = 0.018). It is shown that E-selectin, sLe(x), and MIP-1alpha are synthesized and expressed by epithelial cells, indicating that CAMs are not only involved in leukocyte extravasation and migration, but also in the interaction between leukocytes and colonic epithelium. This knowledge might contribute to the development of improved treatments in UC.     

BB-10010: an active variant of human macrophage inflammatory protein-1 alpha with improved pharmaceutical properties

The stem cell inhibitor, macrophage inflammatory protein-1 alpha (MIP-1 alpha) or LD78, protects multipotent hematopoietic progenitors in murine models from the cytotoxic effects of chemotherapy. Clinical use of human MIP-1 alpha during chemotherapy could therefore lead to faster hematologic recovery and may allow dose intensification. We have also shown that human MIP-1 alpha causes the rapid mobilization of hematopoietic cells, suggesting an additional clinical use in peripheral blood stem cell transplantation. However, the clinical evaluation of human MIP-1 alpha is complicated by its tendency to associate and form high molecular weight polymers. We have produced a variant of rhMIP-1 alpha, BB-10010, carrying a single amino acid substitution of Asp26 > Ala, with a reduced tendency to form large polymers at physiologic pH and ionic strength. This greatly increases its solubility, facilitating its production and clinical formulation. We confirmed the potency of BB-10010 as a human MIP-1 alpha-like agonist in receptor binding, calcium mobilization, inhibition of colony formation, and thymidine suicide assays. The myeloprotective activity of BB-10010 was shown in a murine model of repeated chemotherapy using hydroxyurea. BB-10010 is therefore an ideal variant with which to evaluate the therapeutic potential of recombinant human MIP-1 alpha.