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.     

Regulation of T lymphocyte trafficking into lymph nodes during an immune response by the chemokines macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta

By virtue of their target cell specificity, chemokines have the potential to selectively recruit leukocyte subpopulations into sites of inflammation. Their role in regulation of T lymphocyte traffic into lymph nodes during the development of an immune response has not previously been explored. The sensitization phase of contact hypersensitivity induced by the hapten, dinitrofluorobenzene (DNFB) in the mouse was used as a model of T lymphocyte trafficking in response to antigenic stimulation. Rapid accumulation of CD8+ and CD4+ T cells in the draining lymph nodes was closely associated with strongly enhanced expression of macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta mRNAs and proteins. Mast cells accumulating in the nodes during DNFB sensitization were the predominant source of MIP-1 beta, whereas MIP-1 alpha was expressed by multiple cell types. Neutralization of these chemokines profoundly inhibited T lymphocyte trafficking into lymph nodes and altered the outcome of a subsequent challenge to DNFB. Thus, beta-chemokines regulate T lymphocyte emigration from the circulation into lymph nodes during an immune response and contribute significantly to the immunologic outcome.     

Macrophage inflammatory protein-1 alpha production in lipopolysaccharide-stimulated human adherent blood mononuclear cells is inhibited by the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine

The release of chemokines such as macrophage-inflammatory protein-1 alpha (MIP-1 alpha) from activated macrophages is a crucial step in cell recruitment necessary for establishing local inflammatory responses. To ascertain the importance of the L-arginine/nitric oxide (NO) pathway in LPS-induced MIP-1 alpha release, we stimulated human adherent PBMC with LPS in the presence of the NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA). L-NMMA decreased LPS-induced MIP-1 alpha protein release (45.5% inhibition) and steady state levels of mRNA (48% inhibition) in adherent PBMC. The concentration of L-NMMA for inhibition of MIP-1 alpha release was dependent on the concentration of L-arginine in the cell culture medium, emphasizing the L-arginine-related action of the drug. Most of the MIP-1 alpha release was attributed to the activity of IL-1 and TNF, since coincubation of LPS-stimulated PBMC with IL-1R antagonist and TNF-binding protein abrogated LPS-induced MIP-1 alpha release (by 76.8%). Analysis of cytokine secretion revealed that, in addition to MIP-1 alpha, L-NMMA inhibited the release of mature IL-1 beta (by 70%) and TNF-alpha (by 53%). In contrast, release of macrophage chemoattractant protein-1 was unaffected; IL-10 was augmented (123.4%) by L-NMMA. In the presence of exogenous NO provided by NO donors, LPS-induced MIP-1 alpha release was enhanced. We concluded that endogenous NO acts as a mediator of inflammation. Since IL-10 is a potent anti-inflammatory cytokine, these data also suggest that L-NMMA acts as an anti-inflammatory agent by specifically altering the balance of pro- and anti-inflammatory cytokines released from LPS-stimulated human PBMC.     

Characterization of humoral immune responses induced by immunization with plasmid DNA expressing HIV-1 Nef accessory protein

Mice immunized with plasmid DNA encoding Nef accessory protein of human immunodeficiency virus type 1 developed high levels of anti-Nef antibodies which were maintained for at least 16 months. These antibodies produced in response to Nef-expressing plasmid DNA did not recognize the linear peptides except the long C-terminal peptide for three of the ten sera. With anti-Nef antibodies produced in mice immunized with the protein Nef without any adjuvant, the same restraint epitope binding was found. On the contrary, anti-Nef antibodies from mice immunized with the protein in Freund's adjuvant showed a broader epitope reactivity pattern. Interestingly, the analysis of immunoglobulin isotype profiles of antibodies generated by the different protocols of immunization showed that plasmid DNA immunization induced predominantly IgG2a, whereas immunization with Nef protein, with or without adjuvant, yielded a preponderance of IgG1 antibodies.     

Augmentation and suppression of immune responses to an HIV-1 DNA vaccine by plasmid cytokine/Ig administration

The use of cytokines has shown promise as an approach for amplifying vaccine-elicited immune responses, but the application of these immunomodulatory molecules in this setting has not been systematically explored. In this report we investigate the use of protein- and plasmid-based cytokines to augment immune responses elicited by an HIV-1 gp120 plasmid DNA vaccine (pV1J-gp120) in mice. We demonstrate that immune responses elicited by pV1J-gp120 can be either augmented or suppressed by administration of plasmid cytokines. A dicistronic plasmid expressing both gp120 and IL-2 induced a surprisingly weaker gp120-specific immune response than did the monocistronic pV1J-gp120 plasmid. In contrast, systemic delivery of soluble IL-2/Ig fusion protein following pV1J-gp120 vaccination significantly amplified the gp120-specific immune response as measured by Ab, proliferative, and CTL levels. Administration of plasmid IL-2/Ig had different effects on the DNA vaccine-elicited immune response that depended on the temporal relationship between Ag and cytokine delivery. Injection of plasmid IL-2/Ig either before or coincident with pV1J-gp120 suppressed the gp120-specific immune response, whereas injection of plasmid IL-2/Ig after pV1J-gp120 amplified this immune response. To maximize immune responses elicited by a DNA vaccine, therefore, it appears that the immune system should first be primed with a specific Ag and then amplified with cytokines. The data also show that IL-2/Ig is more effective than native IL-2 as a DNA vaccine adjuvant.     

Acute and relapsing experimental autoimmune encephalomyelitis are regulated by differential expression of the CC chemokines macrophage inflammatory protein-1alpha and monocyte chemotactic protein-1

Myasthenia gravis (MG) patients develop autoantibodies primarily against the acetylcholine receptor in the motor endplate, but also against intracellular striated muscle proteins, notably titin, the giant elastic protein of the myofibrillar cytoskeleton. Titin antibodies have previously been shown to be directed against a single epitope on the molecule, located at the A-band/I-band junction and referred to as the main immunogenic region (MIR) of titin. By using immunofluorescence microscopy on stretched single myofibrils, we now report that approximately 40% of the sera from 18 MG/thymoma patients and 8 late-onset MG patients with thymus atrophy contain antibodies that bind to a more central I-band titin region. This region consists of homologous immunoglobulin domains and is known to be differentially spliced dependent on muscle type. All patients with I-band titin antibodies also had antibodies against the MIR. Although a statistically significant correlation between the occurrence of I-band titin antibodies and MG severity was not apparent, the results could hint at an initial immunoreactivity to titin's MIR, followed by reactivity along the titin molecule in the course of the 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.     

The inflammatory and immune response to mousepox (infectious ectromelia) virus

The ectromelia virus (EV) has been recognized as the etiological agent of a relatively common infection in laboratory mouse colonies around the world, i.e., Europe (including Poland), USA and Asia. Due to widespread use of mice in biomedical research, it is important to study the biology of strains characteristic for a given country. This is particularly significant for the diagnosis, prevention and control ectromelia. In severe epizootics, approximately 90% morbidity is observed within colonies and mortality rate exceeding 70% is observed within 4 to 20 days from the appearance of clinical symptoms. The resistance to lethal infection is mouse strain-dependent. Several inbred strains of mice, including C57BL/6 and AKR are resistant to the lethal effects of EV infection, while others, such as A and BALB/c are susceptible. Recent studies indicate that (1) T lymphocytes, NK cells and interferon (IFN)-dependent host defenses must operate for the expression of resistance, (2) virus-specific T-cell precursors appear earlier in regional lymph nodes of resistant than susceptible mice, and (3) resistance mechanisms are expressed during early stages of infection. Over the past several years, (1) induction of anti-EV cytotoxic CD8+ T lymphocytes (CTL) responses in vivo in the absence of CD4+ (T helper) cells, (2) importance of some cytokines e.g., IFN-gamma in EV clearance at all stages of infection, and (3) induction of nitric oxide (NO) synthase, which is necessary for a substantial antiviral activity of IFN-gamma, have been demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA vaccination using expression vectors carrying FIV structural genes induces immune response against feline immunodeficiency virus

Following inactivated virus vaccination trials, the surface glycoprotein gp120 of the feline immunodeficiency virus (FIV) was considered as one of the determinants for protection. However, several vaccination trials using recombinant Env protein or some peptides failed to induce protection. To understand the role of the gp120 protein in vivo, we vaccinated cats with naked DNA coding for FIV structural proteins gp120 and p10. We analyzed the ability of these vaccinations to induce immune protection and to influence the onset of infection. Injection in cat muscles of expression vectors coding for the FIV gp120 protein induced a humoral response. Cats immunized twice with the gp120 gene showed different patterns after challenge. Two cats were, like the control cats, infected from the second week after infection onwards. The two others maintained a low proviral load with no modification of their antibody pattern. The immune response induced by gp120 DNA injection could control the level of viral replication. This protective-like immune response was not correlated to the humoral response. All the cats immunized with the gp120 gene followed by the p10 gene were infected, like the control cats, from the second week but they developed a complete humoral response against viral proteins after challenge. Furthermore, they showed a sudden but transient drop of the proviral load at 4 weeks after infection. Under these conditions, one injection of the p10 gene after one injection of the gp120 gene was not sufficient to stimulate protection. On the contrary, after a period, it seems to facilitate virus replication.     

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.     

Integrin alpha 2 beta 1 is a positive regulator of collagenase (MMP-1) and collagen alpha 1(I) gene expression

A classical model for studying the effects of extracellular matrix is to culture cells inside a three-dimensional collagen gel. When surrounded by fibrillar collagen, many cell types decrease the production of type I collagen, and the expression of interstitial collagenase (matrix metalloproteinase-1; MMP-1) is simultaneously induced. To study the role of the collagen-binding integrins alpha 1 beta 1 and alpha 2 beta 1 in this process, we used three different osteogenic cell lines with distinct patterns of putative collagen receptors: HOS cells, which express only alpha 1 beta 1 integrin, MG-63 cells, which express only alpha 2 beta 1 integrin, and KHOS-240 cells, which express both. Inside collagen gels, alpha 1 (I) collagen mRNA levels were decreased in HOS and KHOS-240 cells but not in MG-63 cells. In contrast, MMP-1 expression was induced in KHOS-240 and MG-63 cells but not in HOS cells. Transfection of MG-63 cells with alpha 2 integrin cDNA in an antisense orientation reduced the expression level of alpha 2 integrin. These cell clones showed induction and reduction of mRNA levels for MMP-1, respectively. HOS cells normally lacking alpha 2 beta 1 integrin were forced to express it, and this prevented the down-regulation in the levels of alpha 1 (I) collagen mRNA when cells were grown inside collagen gels. The data indicate that the level of MMP-1 expression is regulated by the collagen receptor alpha 2 beta 1 integrin. The down-regulation of collagen alpha 1 (I) is mediated by another receptor. Integrin alpha 2 beta 1 may compete with it and thus be a positive regulator of collagen synthesis.     

Epitope-specific monoclonal antibodies against human C-terminal procollagen alpha1(III)-propeptide

We have generated monoclonal antibodies against recombinant C-terminal human procollagen alpha1(III) propeptide (PIIICP), produced in E. coli in high yields. The monoclonal antibodies were screened for epitope specificity using recombinant truncated PIIICP. Several antibodies were identified which recognized different regions of the PIIICP molecule. The ability of the antibodies to detect PIIICP antigens in human cell line lysates and supernatants was demonstrated. As PIIICP antigens are a key marker of extracellular matrix metabolism, the monoclonal antibodies described here should be of value for clinical and basic research.     

Abnormalities of measles antibody response in human immunodeficiency virus type 1 (HIV-1) infection

The finding that severe measles occurs in immunized as well as nonimmunized human immunodeficiency virus (HIV)-infected individuals suggests that both immunologic memory and the initial response to measles may be impaired by HIV infection. That the initial response is affected was supported by the finding that post-measles immunization titers of HIV-infected babies were significantly lower (p = 0.01) than those of normal babies. Poor immunologic memory was evidenced in HIV-infected children by lower titers than in normal children (p < 0.001) and by a continuing decline in measles antibody that was not arrested by reimmunization. Impaired memory appeared to be associated with defective avidity maturation. HIV-infected babies and infants or children had a significantly lower avidity index (AI) than age-matched normal children (p < 0.01). HIV-infected adults, who were infected with HIV following infection with measles, did not have AI values significantly different from normal adults (p = 0.18) but had significantly greater values than did HIV-infected babies and children (p < 0.01). Thus, in contrast to infants and children who were infected with HIV before measles immunization, the adult immune response to measles was less affected.     

Circulating form of human vascular adhesion protein-1 (VAP-1): increased serum levels in inflammatory liver diseases

Vascular adhesion protein-1 (VAP-1) is a dimeric 170-kDa endothelial transmembrane molecule that under normal conditions is most strongly expressed on the high endothelial venules of peripheral lymph nodes and on hepatic endothelia. It is a glycoprotein that mediates tissue-selective lymphocyte adhesion in a sialic acid-dependent manner. In this study, we report the detection of a soluble form of VAP-1 in circulation. We developed a quantitative sandwich ELISA using novel anti-VAP-1 mAbs and used it to determine the levels of soluble VAP-1 (sVAP-1) in the serum of healthy individuals and in patients with inflammatory diseases. In healthy persons, circulating sVAP-1 concentrations were 49 to 138 ng/ml. Immunoblotting studies revealed that the apparent molecular mass of dimeric sVAP-1 is slightly (approximately 10 kDa) higher than that of transmembrane VAP-1 under nonreducing conditions. In contrast, the electrophoretic mobilities of monomeric sVAP-1 and transmembrane VAP-1 were similar after reduction and boiling. Adhesion assays showed that the circulating sVAP-1 modulates lymphocyte binding to endothelial cells. Inflammation can cause an elevation of serum sVAP-1 levels, because sVAP-1 concentrations in patients with certain liver diseases were two- to fourfold higher than those in normal individuals. In contrast, rheumatoid arthritis and inflammatory bowel diseases were not associated with elevated levels of sVAP-1. These findings indicate that there is a functionally active, soluble form of VAP-1 in circulation and suggest that the serum level of sVAP-1 might be a useful marker of disease activity in inflammatory liver diseases.