Effects of in vivo T lymphocyte subset depletion on mycobacterial infections in mice.

The relative importance of CD4+ and CD8+ T cell subsets in the expression of acquired resistance to systemic infection by Mycobacterium kansasii was determined. T cell subsets were depleted in thymectomized C57BL/6 mice by the intravenous administration of monoclonal antibodies directed against the relevant T cell determinants. Depletion of the CD4+ subset exacerbated the severity of the infection in intravenously challenged mice. This effect was apparent in the first 2 weeks of the infection and persisted throughout the 12 weeks of the study. On the other hand, depletion of the CD8+ cells had no apparent effect on the growth curves. Infections by Mycobacterium tuberculosis Erdman or bacille Calmette-Guérin (BCG) Pasteur were also substantially enhanced by CD4 depletion, but not by the depletion of CD8+ cells. The effect of subset depletion on infections by M. tuberculosis and BCG was examined in both innately susceptible C57BL/6 mice and innately resistant B6D2 mice.

Role of chemokines and cytokines in a reactivation model of arthritis in rats induced by injection with streptococcal cell walls.

Intraarticular injection of streptococcal cell wall (SCW) antigen followed by intravenous challenge results in a T cell-mediated monoarticular arthritis ill female Lewis rats. Initial studies showed that this reactivation response to intravenous SCW antigen is dependent on the presence of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) and that the early phase of swelling is neutrophil-dependent. Neutrophil depletion or passive immunization with antibodies to P-selectin or macrophage inflammatory protein-2 reduced the intensity of ankle edema and the influx of neutrophils. After the first few days, however, the arthritic response is mediated primarily by mononuclear cells. Joint tissues showed up-regulation of mRNA for monocyte chemotactic protein-1 (MCP-1), which could be inhibited in part by anti-IL-4; treatment of rats with antibodies to IL-4 or MCP-1 significantly suppressed development of ankle edema and histopathological evidence of inflammation. Antibodies to interferon-gamma or IL-10 had no effect. Treatment with anti-MCP-1 also suppressed influx of (111)In-labeled T cells into the ankle joint. These data suggest that the late, mononuclear-dependent phase of SCW-induced arthritis in female Lewis rats requires cytokines that up-regulate MCP-1, which in turn may facilitate recruitment and extravasation of mononuclear cells into the joint.

Phylogeny of neuroimmunoregulation: effects of adrenergic and cholinergic agents on the in vitro antibody response of the rainbow trout, Onchorynchus mykiss.

The ability of the nervous system to influence a variety of immune processes has been well established in mammals. There are, however, few studies on how the peripheral nervous system might affect immunity in the lower vertebrates. This study demonstrates that autonomic neurotransmitters (or their analogs) can significantly influence the in vitro induction of antibody-secreting cells in cultures of splenic leukocytes from the rainbow trout. Beta-adrenergic agonists suppress, while alpha-2 adrenergic and cholinergic agonists enhance the in vitro antibody response to the T-independent antigen TNP-LPS. These effects are receptor mediated as evidenced by the action of specific receptor antagonists. These results, taken with the previous findings that the salmonid spleen contains a rich adrenergic innervation, and that chemical sympathectomy results in an enhanced in vivo antibody response, strongly suggest that autonomic neurotransmitters may affect immune function in teleosts via leukocyte receptors for these factors.

The influence of adrenergic and cholinergic agents on the chemiluminescent and mitogenic responses of leukocytes from the rainbow trout, Oncorhynchus mykiss.

In a previous report, it was shown that agonists of cholinergic or alpha-adrenergic receptors enhance, whereas beta-adrenergic agonists suppress, in vitro antibody responses by splenic leukocytes from rainbow trout. The present study addresses the mechanisms by which autonomic neurotransmitters (or their analogs) could affect this antibody response. Possibilities include an influence on accessory cell function, on the clonal proliferation of antigen-stimulated lymphocytes, and/or on the synthesis and secretion of antibody. Epinephrine and selective beta-adrenergic agonists suppressed whereas both alpha and cholinergic receptor agonists enhanced the ability of stimulated pronephric leukocytes (mainly macrophages and neutrophils) to produce reactive oxygen species, suggesting that accessory cells may be a target of these agents in the antibody response. Beta-adrenergic agonists also suppressed the proliferative response of splenic leukocytes to LPS, Con A, and PHA. There was no effect, however, of alpha-adrenergic or cholinergic-receptor agonists on mitogenic responses.

Leukocytes of rainbow trout (Oncorhynchus mykiss) pronephros: cell types producing superoxide anion.

Fish pronephric and blood leukocytes yield a chemiluminescent response (CL) when stimulated appropriately. This response reflects the production of highly reactive oxygen derivatives which contribute to oxygen-dependent killing of targets such as pathogens and parasites. From suspensions of pronephric cells of the Rainbow trout, Oncorhynchus mykiss, we have obtained populations enriched for CL-positive cells. Four bands of cells were obtained using continuous gradients generated with 60% Percoll. The leukocytes of band II showed a very strong PMA-induced CL response, the magnitude of which was several times higher than that observed with equivalent numbers of cells form unseparated pronephric cell suspensions. Cells present in other bands were not significantly chemiluminescent. Flow cytometric analysis showed that band II contained large granular cells and small granular cells. Cytochemical analysis showed that this subpopulation was greatly enriched with neutrophils. Many band II cells adhere to glass whereas few band III cells do so. The glass-adherent cells loose their CL potential after a few days in vitro, whereas the nonadherent cells retain their CL responsiveness for at least a week in vitro.

The Independent Living Program: an alternative to institutionalization.

The Independent Living Program is a joint venture demonstration project in Ardmore, Pennsylvania. Partners are Community Health Affiliates, a one-hundred-plus-year-old visiting nurse association and integrated home care agency, and neighboring Ardmore House, a 63-unit residential facility for a well-elderly population with extremely limited financial means.

Autonomic innervation of the spleen of the coho salmon, Oncorhynchus kisutch. a histochemical demonstration and preliminary assessment of its immunoregulatory role.

Regulation of immunity by the nervous system, now a well-established phenomenon in mammals, is effected in part through the autonomic innervation of lymphoid tissues. Noradrenergic fibers specifically target lymphocyte-rich areas in mammalian lymphoid tissues, and their ablation, or the administration of adrenergic agents, can significantly alter immune responses. This study demonstrates that the spleen of the coho salmon is also richly innervated by adrenergic neurons. While this innervation enters the spleen and remains largely associated with the splenic vasculature, fibers can also be observed entering the parenchyma. Although the coho spleen does not possess a well-developed white pulp, aggregations of leukocytes are found adjacent to the major blood vessels in close proximity to the vascular nervous tissue and parenchymal fibers. Chemical sympathectomy with 6-hydroxydopamine results in a significant enhancement of the splenic antibody-secreting cell response to trinitrophenylated sheep red blood cells. These results suggest that sympathectomy is removing a constraint, in the form of inhibitory catecholamines, on the immune response. The potential benefits from a teleost model of neural-immune interactions are discussed.

Pulmonary granuloma formation in the rat is partially dependent on monocyte chemoattractant protein 1.

BACKGROUND: We have examined the role of MCP-1 (monocyte chemoattractant protein 1; also known as monocyte chemotactic and activating factor or the murine JE gene product) in the pathogenesis of glucan-induced granulomatous vasculitis in the rat. While in vitro studies indicate that MCP-1 possesses monocyte chemotactic and activating activities, little is known about its biologic role in pathologic processes. Glucan-induced pulmonary granulomatous vasculitis is an ideal model in which to study the role of MCP-1, because the granulomas develop rapidly and synchronously and are monocyte/macrophage-rich. EXPERIMENTAL DESIGN: The purpose of this study was to define the topographic distribution and temporal pattern of MCP-1 expression in the lungs of rats with evolving glucan-induced granulomatous vasculitis and to determine the effect of neutralization of MCP-1 activity on granuloma formation. Glucan-induced pulmonary granulomatous vasculitis was induced in rats by the intravenous infusion of yeast cell wall glucan. At the indicated time points after glucan infusion, rats were sacrificed and the lungs processed for Northern, immunohistochemical and in situ hybridization analyses of MCP-1 production. Morphometric analysis was used to quantify the effect of neutralization of MCP-1 activity on granuloma formation. RESULTS: Granuloma formation was accompanied by a biphasic increase in steady-state whole lung MCP-1 mRNA levels that peaked at 1 and 6 to 24 hours. In situ hybridization and immunohistochemical analyses revealed that components of the bronchial and vascular walls are responsible for the early rise (1 hour) in MCP-1 mRNA and protein expression, whereas granuloma-associated alveolar macrophages are the predominant source of MCP-1 later (6 to 24 hours) in the evolution of these lesions. Intravenous infusion and/or intratracheal instillation of neutralizing concentrations of anti-rat MCP-1 antibody raised against recombinant rat MCP-1 resulted in a dramatic decrease in the number and size of glucan-induced granulomas as well as in the numbers of mononuclear phagocytes retrieved in bronchoalveolar lavage fluid. CONCLUSIONS: These studies demonstrate that glucan-induced granulomatous vasculitis is accompanied by increased local expression of MCP-1 mRNA and protein, that there is a coordinated production of MCP-1 by different cell types within the lung during evolving glucan-induced pulmonary vasculitis, and that MCP-1 plays a requisite role in pulmonary granuloma formation.

Analysis of monocyte chemoattractant protein 1-mediated lung injury using rat lung organ cultures.

Using a rat lung organ culture system, we analyzed the role of monocyte chemoattractant protein 1 (MCP 1) in leukocyte to lung adhesive interactions and monocyte-mediated lung injury. Quantitative leukocyte to lung adhesive interactions were examined using an adaptation of the Woodruff-Stamper frozen section binding assay. Pretreatment of organ cultures with recombinant human tumor necrosis factor (rhTNF alpha) resulted in a protein synthesis-dependent increase in the adhesiveness of lung tissue for peripheral blood monocytes. Adhesion of monocytes to lung tissue was not increased above baseline after 7 hours but increased more than twofold by 24 hours and persisted through 48 hours. Binding of monocyte to lung tissue was further increased when recombinant rat MCP 1 was added to monocyte suspensions immediately before being layered onto lung sections derived from either TNF alpha-treated or untreated organ cultures. Addition of antibody directed against rat CD11b/c resulted in a moderate reduction in monocyte binding. TNF or lipopolysaccharide-induced activation of mononuclear cells in the presence of [3H]leucine-labeled organ cultures resulted in lung injury as assessed by radioisotope release. Mononuclear cell-mediated organ culture injury could be partially inhibited with anti-rat MCP 1 antibody, anti-rat CD11b/c antibody, or antioxidants including catalase and deferoxamine. Anti-MCP 1 and anti-CD11b/c increased the absolute numbers of monocytes that could be retrieved from monocyte-lung co-cultures while catalase and deferoxamine did not. In vitro studies revealed that isolated rat peripheral blood monocytes produce O2- in response to MCP 1. These data provide a functional correlate for recent in vitro studies which suggest that MCP 1 may mediate leukocyte adhesive processes by up-regulating beta 2 integrin expression on monocytes. This study provides evidence that monocytes activated by MCP 1 can damage lung tissue through an oxidant-mediated mechanism. Monocyte chemoattractant protein 1 may participate in the pathogenesis of monocyte-mediated lung injury by modulating inflammatory cell adhesion as well as through monocyte activation.

Immunization of mice with mycobacterial culture filtrate proteins.

Culture filtrate proteins were obtained from Mycobacterium tuberculosis cultures after 7 days growth in Proskauer and Beck medium. The protein yield increased substantially to peak about the time the number of viable organisms reached its maximum level (day 8). Examination of the protein concentrate by SDS-PAGE revealed the presence of at least 12 separate protein bands varying from 10 to 90 kD. Mice were injected subcutaneously with 20 micrograms of M. tuberculosis culture filtrate (MTCF) protein suspended in saline or Freund's complete or incomplete adjuvant. The vaccinated mice were subjected to an aerogenic challenge with 10(3) colony-forming unit (CFU) M. tuberculosis Erdman and a significant reduction in the number of viable organisms was observed in the spleens and lungs determined over a 21-day period compared with age-matched normal controls. Mice immunized with the same culture filtrate proteins bound to nitrocellulose particles also showed some resistance to the virulent challenge, suggesting that individual antigens present in the culture filtrate were able to induce a protective T cell-mediated immune response in appropriately immunized mice.

T-cell immune responses in Mycobacterium avium-infected mice.

Mycobacterium avium infection was substantially more severe in C57BL/6 (Bcgs) than in (C57BL/6 x DBA/2)F1 hybrid (Bcgr) mice both in terms of bacterial growth in the spleens and lungs and in host survival. Prior Mycobacterium bovis BCG vaccination resulted in increased resistance as well as enhanced tuberculin hypersensitivity to both PPD-S (Mycobacterium tuberculosis) and PPD-A (M. avium). Mice heavily infected with M. avium were used as T-cell donors in an adoptive transfer system. Substantial resistance was observed for both recipient hosts regardless of the genotype of the donor strain. Transfer of resistance was ablated by treatment of the immune spleen cells with anti-Thy 1.2 monoclonal antibody and complement or by cyclophosphamide treatment. Spleen cells which were monodepleted of L3T4+ or Lyt-2+ T cells did not lose their ability to transfer resistance against a subsequent challenge. However, when these cells were doubly deleted, all resistance was ablated in both the BCG-susceptible and -resistant mice. The recipient host expressed a detectable adoptive immune response although the donor had been unable to reduce the growth of the primary M. avium infection in vivo.

Memory T cell-mediated resistance to Mycobacterium tuberculosis infection in innately susceptible and resistant mice.

The memory T cell immune response to Mycobacterium tuberculosis infection was examined in strains of mice which vary in their natural susceptibility to Mycobacterium bovis BCG infection. Naturally susceptible (NS) C57BL/6 and naturally resistant (NR) B6D2 F1 hybrid mice were infected with a sublethal dose of M. tuberculosis and then given antibiotic therapy beginning 2 weeks postinfection. T cells from both strains of mice transferred significant levels of resistance to syngeneic mice challenged aerogenically with M. tuberculosis. This memory response was not substantially reduced by depletion of either L3T4+ or Lyt2+ T cells from the donor mice but was ablated by depletion of both T cell subsets. Cyclophosphamide pretreatment of C57BL/6 memory T cell donors also ablated the resistance transferred to recipient mice. In contrast, B6D2 memory T cells were not affected by cyclophosphamide treatment, suggesting that differences may exist in the metabolic state of the memory T cells in the two donor strains, despite the fact that they both develop similar levels of acquired resistance to a subsequent tuberculous challenge.

Immunization of mice with the antigen A60 of Mycobacterium bovis BCG.

Antigen 60 (A60) is a thermostable component of the cytoplasm of Mycobacterium tuberculosis and BCG which can be fractionated into at least 15 protein bands when analysed by Western blot. Normal B6D2 mice were immunized subcutaneously with 20 micrograms of the A60 protein suspended in Freund's incomplete adjuvant (FIA) or in saline. Three weeks later the mice received a second dose of vaccine followed 2 weeks later by an aerogenic challenge with approximately 10(3) CFU of M. tuberculosis Erdman. The mice receiving the adjuvanted A60 showed a significant reduction (P less than 0.05) in the number of viable organisms recovered from the lungs and the spleen 3 weeks after challenge. However, this response was less than that seen in BCG vaccinated controls.

Regulatory roles of tumor necrosis factor-alpha and interleukin-1 beta in monocyte chemoattractant protein-1-mediated pulmonary granuloma formation in the rat.

Intravenous infusion of particulate yeast cell wall glucan into rats results in the synchronous development of angiocentric pulmonary granulomas that are composed almost entirely of monocytes and macrophages. Previous studies indicate that locally produced monocyte chemoattractant protein-1 (MCP-1) is required for full granuloma development. Because tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 (IL-1) can induce MCP-1 production in a variety of cell types, we sought to determine their potential regulatory roles in this model. A single infusion of anti-TNF-alpha antibody at the time of glucan infusion (time 0) markedly reduced MCP-1 mRNA levels at 1 and 6 hours but not at later time points; there was no effect on granuloma size or number measured at 48 hours. When multiple infusions of anti-TNF-alpha antibody were administered over a 23-hour period (0 to 23 hours), MCP-1 mRNA was reduced through 24 hours, there was a significant reduction in peak bronchoalveolar lavage fluid MCP-1 activity at 48 hours, and there were marked reductions in granuloma size and number at 48 hours. Similar results were observed in animals that received infusions of anti-IL-1 beta. Infusion of anti-IL-1 beta at time 0 resulted in moderate reductions in MCP-1 mRNA at 1 and 6 hours and had no effect on granuloma size or number measured at 48 hours. When multiple infusions of anti-IL-1 beta were administered over a 23-hour period (0 to 23 hours), MCP-1 mRNA was reduced through 24 hours, there was a moderate reduction in peak bronchoalveolar lavage fluid MCP-1 activity at 48 hours, and there were marked reductions in granuloma size and number at 48 hours. A single infusion of anti-TNF-alpha and anti-IL-1 beta together at time 0 resulted in marked reductions in whole lung MCP-1 and mRNA at 1 and 6 hours, but not at 24 hours. Multiple combined infusions of anti-TNF-alpha and anti-IL-1 beta over a 23-hour period resulted in additive reductions in MCP-1 mRNA through 24 hours, bronchoalveolar lavage fluid MCP-1 activity at 48 hours, and granuloma size and number at 48 hours. These data suggest that locally produced TNF-alpha and IL-1 beta play regulatory roles in glucan-induced pulmonary granulomatous vasculitis through the modulation of local MCP-1 production.

Role of intercellular adhesion molecule-1 in glucan-induced pulmonary granulomatosis in the rat.

Glucan-induced pulmonary granulomatous vasculitis in the rat mimics several human lung diseases (e.g., Wegener's granulomatosis, intravenous talcosis). We sought to clarify the role of intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of glucan-induced granulomatous vasculitis. Immunohistochemical analysis of lung sections from rats with florid vasculitis (48 hours) revealed marked alveolar septal and lesional expression of ICAM-1. An ex vivo binding analysis with isotope-labeled antibodies and lung sections taken at various times up to 48 hours after glucan infusion revealed a progressive increase in whole-lung ICAM-1 expression. In vivo measurements of vascular wall-associated ICAM-1 expression revealed an earlier rise that began less than 6 hours after glucan infusion, peaked at 24 to 48 hours, and then declined to near baseline during the ensuing 24 to 96 hours. To assess whether ICAM-1 expression both within blood vessel walls and within lesions per se is important in granuloma development, we carried out in vivo neutralization experiments with several different routes of administration of antibody to ICAM-1. Monoclonal antibody to rat ICAM-1 was either infused intravenously at time 0 (when glucan was infused), infused intravenously at time 0 and after 24 hours, instilled only intratracheally 24 hours after glucan infusion, or given both intravenously (time = 0 and 24 hours) and intratracheally (time = 24 hours). Infusions of monoclonal antibody to rat ICAM-1 resulted in dose-dependent reductions in mean granuloma number and cross-sectional area. Intrapulmonary instillation of antibody to rat ICAM-1 (via tracheostomy 24 hours after glucan infusion) resulted in a modest reduction in mean granuloma number and cross-sectional area. When antibody to ICAM-1 was both infused and instilled via the trachea, we found an additive reduction in mean granuloma size and number. There was a 12-fold increase in adhesion of ED-1-positive peripheral blood mononuclear cells (monocytes) to granuloma-bearing frozen lung sections prepared 48 hours after glucan infusion. Moreover, 73% of the additional adherent monocytes were bound specifically to granulomas per se. The increase in ex vivo monocyte binding to lung sections prepared at 48 hours was reduced 62% when sections were incubated with monoclonal antibody to ICAM-1. Taken together, these data indicate that ICAM-1 expression in evolving glucan-induced granulomatous vasculitis occurs first within blood vessel walls and then within lesional cells per se. The in vivo blocking studies suggest that ICAM-1 expression in both anatomic sites is important in granuloma development.

Expression of monocyte chemoattractant protein-1 in the corpus luteum of the rat.

The known accumulation of macrophages in corpora lutea (CL) at the time of luteal regression prompted us to investigate whether the chemoattractant protein monocyte chemoattractant protein-1 (MCP-1) is expressed in the rat CL. On the day of confirmed mating (Day 0 of pregnancy), regressing CL from the previous (nonfertile) estrous cycle contained immunodetectable MCP-1 and numerous monocytes/macrophages, whereas the newly formed CL of pregnancy, within the same ovary, contained little MCP-1 and few monocytes/macrophages. MCP-1 diminished in the regressing CL on Days 3 and 9 of pregnancy, although numerous monocytes/macrophages remained. The CL of pregnancy on Days 3 and 9 of pregnancy contained minimal MCP-1 and relatively few monocytes/macrophages. By Days 17 and 21 of pregnancy, however, prior to parturition and prior to an accumulation of monocytes/macrophages, expression of MCP-1 increased in the CL of pregnancy. Northern blots revealed a resurgence of luteal MCP-1 mRNA on Day 21 of pregnancy: 3805 +/- 1077 on Day 21 vs. 1059 +/- 177 on Day 9 (p < 0.05; expressed as densitometric units relative to beta-actin). In conclusion, the expression of MCP-1 in the rat CL in association with, or preceding, the appearance of monocytes/macrophages at the time of luteal regression is consistent with the known role of MCP-1 as a potent chemoattractant for monocytes/macrophages. This suggests that MCP-1 might have a prominent role in the immunological process of luteal regression.

The effects of the phospholipase A2 inhibitor, manoalide, on cartilage degradation, stromelysin expression, and synovial fluid cell count induced by intraarticular injection of human recombinant interleukin-1 alpha in the rabbit.

OBJECTIVE: To evaluate the effects of the phospholipase A2 (PLA2) inhibitor manoalide on cartilage degradation, stromelysin expression, and inflammatory cell accumulation in rabbits treated intraarticularly with recombinant human interleukin-1 alpha (rHuIL-1 alpha). METHODS: Rabbits were given an intraarticular injection of rHuIL-1 alpha. At various time points over a 24-hour period, the rabbits were euthanized and the articular space was lavaged with sterile PBS. The proteoglycan content of the lavage fluid was measured using a dimethylmethylene blue assay. PLA2 activity and differential cell counts were also measured. The femur was removed and cartilage proteoglycan content determined. In some experiments, levels of synovial stromelysin messenger RNA (mRNA) were assessed. Manoalide or vehicle was administered 30 minutes before the rHuIL-1 alpha injection. RESULTS: The rHuIL-1 alpha-induced arthritic response is characterized by significant accumulation of inflammatory cells, loss of proteoglycan from the condylar cartilage, and induction of mRNA for stromelysin. PLA2 activity was also elevated in synovial fluids from rHuIL-1 alpha-injected joints. Pretreatment with manoalide (0.3 mg/joint) significantly inhibited PLA2 activity in the synovial fluid, prevented the loss of proteoglycan from the condylar cartilage, and reduced proteoglycan levels in lavage fluids. However, manoalide either had no effect on, or stimulated, cell accumulation. To assess the relationship between the induction of PLA2 and stromelysin, levels of stromelysin mRNA were measured in synovial tissue from manoalide- and vehicle-treated joints. Stromelysin message levels were significantly suppressed in a dose-dependent manner. CONCLUSION: These studies demonstrate that manoalide is a potent inhibitor of inflammation and cartilage catabolism, and suggest that PLA2 is involved in the pathophysiology of rHuIL-1 alpha-induced arthritis in rabbits.

Potential role of monocyte chemoattractant protein 1/JE in monocyte/macrophage-dependent IgA immune complex alveolitis in the rat.

We have examined the role of monocyte chemoattractant protein 1 (MCP 1) in the pathogenesis of monocyte/macrophage-dependent IgA immune complex alveolitis in the rat. Rat MCP 1 was cloned and expressed in order to facilitate analysis of its function in rat models of human disease. A cDNA library was constructed from rat pulmonary artery endothelial cells stimulated with TNF-alpha. The cDNA library was screened with synthetic oligonucleotide probes based on the recently published rat MCP 1 cDNA sequence. Among numerous MCP 1-positive clones, four full length (approximately 480 bp) cDNA were rescued, amplified by polymerase chain reaction, and ligated into a pJVETLZ baculovirus transfer vector. Spodoptera frugiperda insect cells (Sf-21) infected with baculovirus recombinants (Auto-grapha california nuclear polyhedrosis virus) bearing properly oriented MCP 1 cDNA (AcMCP 1) directed the expression of unique peptides of 18, 21, and 23 kDa. Treatment of AcMCP 1-infected Sf-21 cells with tunicamycin resulted in reduced production of the 21- and 23-kDa proteins and an increase in 16- to 18-kDa products, the predicted size range of uncleaved and nonglycosylated rat MCP 1. Denatured and refolded 23-kDa and 21-kDa rat MCP 1 species exhibited dose-dependent monocyte-specific chemotactic activity at concentrations as low as 10(-10) M whereas the 18-kDa species exhibited negligible activity. Antibodies that react with the immunoblot, block rat rMCP 1-directed monocyte chemotaxis, and neutralize monocyte-specific chemotactic activity secreted by TNF-stimulated rat endothelial cells were raised in rabbits immunized with the 23-kDa MCP 1 species. Intravenous administration of anti-MCP 1 antibodies upon initiation of IgA immune complex lung injury resulted in a marked reduction in lung injury as measured by pulmonary vascular permeability, alveolar hemorrhage, and pulmonary monocyte/macrophage recruitment and pulmonary monocyte/macrophage recruitment. These data suggest that MCP 1 may play an important role in the pathogenesis of monocyte/macrophage-dependent IgA immune complex alveolitis in the rat.

The membrane attack complex of complement induces interleukin-8 and monocyte chemoattractant protein-1 secretion from human umbilical vein endothelial cells.

Cell surface assembly of the membrane attack complex (MAC) of complement occurs in a variety of pathophysiological settings. Depending upon the density and size distribution of pores formed by the MAC and the functional integrity of membrane regulators of complement activation, the MAC can either cause direct cell lysis or transduce cell activation. We have examined the functional capacity of sublytic concentrations of MAC to induce the secretion of specific alpha- and beta-chemokines from human umbilical vein endothelial cells (HUVECs). Endothelial cell activation by the MAC has particular relevance to complement-dependent inflammatory processes including ischemia-reperfusion injury and acute lung injury. Assembly of sublytic concentrations of the MAC on HUVECs resulted in the sequential secretion of both neutrophil and monocyte chemotactic activities. Analysis of conditioned medium from MAC-bearing HUVECs revealed that the neutrophil chemotactic activity was largely attributable to interleukin (IL)-8, whereas the monocyte chemotactic activity, which was detected later (peak at 8 hours versus 4 hours), was largely attributable to MCP-1. This temporal pattern of MAC-induced secretion of IL-8 and MCP-1 was confirmed using IL-8- and MCP-1-specific enzyme-linked immunosorbent assays. Northern hybridization analysis of HUVECs revealed that MAC deposition was accompanied by an increase in IL-8 and MCP-1 mRNA levels. These data indicate that assembly of sublytic concentrations of the MAC on HUVECs can induce the sequential secretion of both neutrophil and monocyte chemotactic activities and that the former is largely attributable to IL-8 whereas the latter is largely attributable to MCP-1.

Expression of monocyte chemoattractant protein-1 (MCP-1) by rat alveolar macrophages during chronic lung injury.

Using a well-characterized model of bleomycin-induced pulmonary fibrosis in the rat, we determined that there was a time-dependent elaboration of monocyte chemotactic activity in bronchoalveolar lavage fluid. Northern hybridization analysis revealed markedly increased expression of rat monocyte chemoattractant protein-1 (MCP-1) mRNA in alveolar macrophages (AMs) from rats following induction of pulmonary fibrosis. Monocyte chemotactic activity was also significantly increased in conditioned media from AMs retrieved from injured rat lungs. These data suggest that one important role of AMs in the pathogenesis of chronic inflammatory lung injury and pulmonary fibrosis is the regulation of monocyte recruitment and activation within the lung secondary to secretion of monocyte chemoattractants including MCP-1.