Complement Receptors CR1 Research Articles

Regulation of B-cell activation by complement receptors CD21 and CD35.

The role of complement in the development and regulation of antibody responses under both healthy and pathological conditions is known for long. Unraveling the elements involved and the molecular mechanisms underlying the events however is still in progress. This review focuses on the role of complement receptors CR1 (CD35) and CR2 (CD21) expressed on B lymphocytes, which interact with ligands generated upon activation of component C3, the major protein of the complement cascade. The binding and possible effects of immune complexes comprising antigen, antibody and complement on B-cell activation are discussed. Results of clinical studies of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis and conclusions drawn from animal models used to investigate various aspects of human diseases are also debated. We discuss similarities regarding the overall structure and certain functions of complement and complement receptors in mice and men however, call the attention to major differences regarding tissue distribution and their role in B-cell functions.

Insulin-like growth factor binding protein-2 modulates podocyte mitogenesis.

To study the role of insulin-like growth factors (IGF) in podocyte maturation, we isolated and characterized fetal visceral glomerular epithelial cells from human kidneys obtained at 8-18 weeks gestation. Cells were identified as podocyte lineage by their cobblestone morphology and immunoreactivity with synaptopodin, Wilms tumor-1 suppressor gene product (WT-1), complement receptor CR1, and cytoskeletal proteins smooth muscle actin and vimentin. Stimulation of the podocyte cell monolayers with IGF-II resulted in a slight increase in mitogenesis, an effect that was concentration and time dependent and abrogated by co-incubation with exogenous IGF binding protein 2 (IGFBP-2). Western blot analysis of conditioned media revealed that cultured podocytes expressed endogenous IGFBP-2 exclusively. IGF-II stimulation enhanced IGFBP-2 production in a dose- and time-dependent fashion and was associated with an increase in IGFBP-2 mRNA production. These data demonstrate that IGF-II-stimulated IGFBP-2 production appears to inhibit the mitogenic effect of IGF-II, and may have an autocrine effect on the maturation, differentiation, and survival of fetal podocytes.

Mycobacterium tuberculosis-infected human macrophages exhibit enhanced cellular adhesion with increased expression of LFA-1 and ICAM-1 and reduced expression and/or function of complement receptors, FcgammaRII and the mannose receptor.

The entry of Mycobacterium tuberculosis (Mtb) into the host macrophage and its survival in this environment are key components of tuberculosis pathogenesis. Following intracellular replication of the bacterium within alveolar macrophages, there is spread of bacilli to regional lymph nodes in the lungs and subsequent presentation of antigens to the host immune system. How this process occurs remains poorly understood, but one mechanism may involve the migration of macrophages containing Mtb across the alveoli to lymph nodes, where there is development of a protective host response with formation of granulomas composed in part of aggregated and fused, apoptotic, infected macrophages. Leukocyte integrins, including lymphocyte function-associated antigen-1 (LFA-1) and complement receptors CR3 and CR4, and their counter receptors play a major role in macrophage adhesion processes and phagocytosis. In this study, the appearance of Mtb-infected macrophages over time was examined, using inverted-phase microscopy and an in vitro culture model of human monocyte-derived macrophages (MDMs). Prior to and immediately following infection of the MDMs with Mtb, the macrophages appeared as individual cells in monolayer culture; however, within 24 h of infection with Mtb, the MDMs began to migrate and adhere to each other. The kinetics of this response were dependent on both the m.o.i. and the length of infection. Quantitative transmission electron microscopy studies revealed that macrophage adhesion was accompanied by increases in levels of LFA-1 and its counter receptor (ICAM-1), decreases in surface levels of the phagocytic receptors CR3, CR4 and FcgammaRII, and an increase in major histocompatibility complex Class II (MHC-II) molecules at 72 h post-infection. Decreases in surface levels of CR3 and CR4 had a functional correlate, with macrophages containing live bacilli showing a diminished phagocytic capacity for complement-opsonized sheep erythrocytes; macrophages containing heat-killed bacilli did not show this diminished capacity. The modulation of macrophage adhesion and phagocytic proteins may influence the trafficking of Mtb-infected macrophages within the host, with increases in levels of LFA-1 and ICAM-1 enhancing the adhesive properties of the macrophage and decreases in phagocytic receptors diminishing the phagocytic capacity of an already-infected cell, potentially allowing for maintenance of the intracellular niche of Mtb.

Complement component C3 promotes T-cell priming and lung migration to control acute influenza virus infection.

The complement cascade defines an important link between the innate and the specific immune system. Here we show that mice deficient for the third component of complement (C3-/- mice) are highly susceptible to primary infection with influenza virus. C3-/- mice showed delayed viral clearance and increased viral titers in lung, whereas mice deficient for complement receptors CR1 and CR2 (Cr2-/- mice) cleared the infection normally. Priming of T-helper cells and cytotoxic T cells (CTLs) in lung-draining lymph nodes was reduced, and the recruitment into the lung of virus-specific CD4+ and CD8+ effector T cells producing interferon-gamma was severely impaired in C3-/- but not in Cr2-/- mice. Consequently, T-helper cell-dependent IgG responses were reduced in C3-/- mice but remained intact in Cr2-/- mice. These results demonstrate that complement induces specific immunity by promoting T-cell responses.

Complements for mad-cow disease

BSE, scrapie and new variant Creutzfeldt–Jakob disease are caused by exposure to pathological forms of prion protein (PrPSc). Following amplification in the periphery, PrPSc invades the nervous system by an as-yet-unknown mechanism. Follicular dendritic cells (FDCs) are the major site of PrPSc accumulation outside of the CNS. Indeed, FDCs play a pivotal role in disease progression, as animals lacking FDCs are protected from disease. Under physiological conditions, FDCs ‘fish’ Ags from the circulation and store them in their native form on the cell surface; these trapped Ags are presented to specific B cells for the initiation and maintenance of Ab responses. This Ag-trapping mechanism might occur through Fc receptors or the complement receptors CD21 and/or CD35. Thus, a functional complement cascade and/or Ab system is required for the efficient capture of Ag by FDCs.Two articles by Mabbott et al. 1xTemporary depletion of complement component C3 or genetic deficiency of C1q significantly delays onset of scrapie. Mabbott, N.A. et al. Nat. Med. 2001; 7: 485–487Crossref | PubMed | Scopus (176)See all References1 and Klein et al. 2xComplement facilitates early prion pathogenesis. Klein, M.A. et al. Nat. Med. 2001; 7: 488–492Crossref | PubMed | Scopus (247)See all References2 assess the role of complement and Fc receptors in the progression of murine scrapie. The absence of C3, a crucial component of both the classical and alternative pathways of the complement cascade, significantly delayed disease progression and reduced the burden of PrPSc in the periphery. A deficiency of complement receptors CR1 and CR2 (CD21 and CD35, respectively) had a similar impact on disease progression, suggesting that C3 is responsible for the deposition of PrPSc through complement receptors. Interestingly, the absence of PrPSc-specific Abs and a variety of Fc receptors (FcγRI–FcγRIII) had no influence on the disease. This is consistent with the observation that no anti-PrPSc Abs can be detected in infected animals, probably because the host's immune system remains unresponsive to PrPSc. Nevertheless, it remains possible that the combination of a deficiency in Fc receptors and complement might result in a further reduction in the replication of PrPSc. This is in line with the observation that the requirement for complement for disease progression is not absolute, as high doses of PrPSc were able to induce scrapie with normal kinetics in complement-deficient mice. This is reminiscent of some anti-viral B-cell responses, where complement-dependency can be overcome by high Ag-load.These observations demonstrate a pivotal, albeit not absolute, role for complement in the induction of scrapie, and suggest that temporal elimination of components of the complement cascade might be a potential therapy for infected individuals. The studies also make an interesting case for how infectious agents, including, in this case, the simplest-known agent, can exploit the properties of the immune system for their own purposes. FDCs store native Ags and present them to B cells; to perform this task, they have evolved receptors that bind foreign Ags and mechanisms that protect the Ags from degradation and phagocytosis by other cell types. It is precisely this property of FDCs that is exploited by the scrapie-agent and allows PrPSc not only to persist safely, but even to replicate, within the heart of the immune system. It is hard to believe that all this is achieved by a protein that apparently does not even carry any genetic information!

Genetics of human complement component C4 and evolution the central MHC

The two classes of human complement component C4 proteins C4A and C4B manifest differential chemical reactivities and binding affinities towards target surfaces and complement receptor CR1. There are multiple, polymorphic allotypes of C4A and C4B proteins. A complex multiplication pattern of C4A and C4B genes with variations in gene size, gene dosage and flanking genes exists in the population. This is probably driven by the selection pressure to respond to a great variety of parasites efficiently and effectively, which the bony fish achieved through the multiplication and diversification of the related complement C3 proteins. Complement C4, C3 and C5 belong to the alpha2 macroglobulin protein family but acquired specific features that include an anaphylatoxin domain, a netrin (NTR) domain, and stretches of basic residues for proteolytic processings to form multiple chain structures. Complement C3 and C4 are important in the innate immune response as they opsonize parasites for phagocytosis. The emergence of complement C3 predates proteins involved in the adaptive immune response as C3 is present in deuterostome invertebrates such as echinoderms. The human C4 genes are located in the central MHC at chromosome 6p21.3. C3 and C5 are located at chromosome 19 and 9, respectively, with representatives of the other groups of genes paralogous to the MHC at 19p13.1-p13.3, 1q21-25, and 9q33-34. The central MHC also contains genes for complement components C2 and Bf. These genes appear to have similar evolutionary histories to C3/C4/C5 and are used here to illustrate stepwise processes resulting in co-location of diverse domains, chromosomal duplication, local segmental duplication and divergence of sequence and function. This model of evolution is useful in the investigation of innate and acquired immunity and in seeking explanations for diseases associated with MHC ancestral haplotypes.

Complement C4 inhibits systemic autoimmunity through a mechanism independent of complement receptors CR1 and CR2.

The complement system enhances antibody responses to T-dependent antigens, but paradoxically, deficiencies in C1 and C4 are strongly linked to autoantibody production in humans. In mice, disruption of the C1qa gene also results in spontaneous autoimmunity. Moreover, deficiencies in C4 or complement receptors 1 and 2 (CR1/CR2) lead to reduced selection against autoreactive B cells and impaired humoral responses. These observations suggest that C1 and C4 act through CR1/CR2 to enhance humoral immunity and somehow suppress autoimmunity. Here we report high titers of spontaneous antinuclear antibody (ANA) in C4(-/)- mice. This systemic lupus erythematosus-like autoimmunity is highly penetrant; by 10 mo of age, all C4(-)(/)- females and most males produced ANA. In contrast, titers and frequencies of ANA in Cr2(-)(/)- mice, which are deficient in CR1 and CR2, never rose significantly above those in normal controls. Glomerular deposition of immune complexes (ICs), glomerulonephritis, and splenomegaly were observed in C4(-)(/)- but not Cr2(-)(/)- mice. C4(-)(/)-, but not Cr2(-)(/)-, mice accumulate activated T and B cells. Clearance of circulating ICs is impaired in preautoimmune C4(-)(/)-, but not Cr2(-)(/)-, mice. C4 deficiency causes spontaneous, lupus-like autoimmunity through a mechanism that is independent of CR1/CR2.

Early microglial reaction following mild forebrain ischemia induced by common carotid artery occlusion in rats

Early microglial reaction following mild ischemic injury caused by bilateral common carotid artery occlusion has been investigated in rats. The ischemic insults lasted for 10, 15 and 20 min without recirculation, and with several reperfusion intervals from 1 h to 3 days. The resting and activated microglial cells were visualized with immunohistochemistry using monoclonal antibodies raised against the CR3 complement receptor, the MHC class I and class II antigens, the macrophage common antigen and with Bandeiiraea simplicifolia lectin-histochemistry. The neuroprotective effect of hypothermia on the early microglial activation was also studied. Ten minutes bilateral common carotid artery occlusion in hypothermic rats without reperfusion caused a mild microglial reaction in the hippocampus. Strong reaction was seen following 20 min insult without reperfusion. Ischemia followed by recirculation caused milder reaction than without reperfusion. Our results suggest that the microglial cells are very sensitive indicators of a mild, transient ischemic insult that does not result in neuronal cell death.

Effects of polysaccharides from Silene vulgaris on phagocytes

The effects of the polysaccharides isolated from the intact plant (pectic polysaccharides P1, P2 and P3) and from the callus (acidic arabinogalactan C1 and pectin C2) of Silene vulgaris on phagocytic activity were studied in relation to an uptaking capacity and a myeloperoxidase activity of the peripheral human neutrophils and monocytes and rat peritoneal macrophages in vitro. Both intact plant and callus polysaccharides were shown to increase uptaking capacity of peripheral phagocytes. The callus acidic arabinogalactan C1 was only found to stimulate lysosomal activity of the peripheral phagocytes. Some polysaccharides studied were established to effect on peritoneal resident macrophages. Pectins P1, P3 and C2 failed to enhance myeloperoxidase activity of the macrophages in calcium-free solution, whereas the effect of callus arabinogalactan C1 was established to be independent of extracellular calcium. Polysaccharides studied failed to influence neither complement receptor CR3- nor scavenger receptor SR-mediated adhesion of the macrophages. The data obtained demonstrate that the intact S. vulgaris and its callus may be used as sources of immunoactive polysaccharides and that pectins and weakly acidic arabinogalactan seemed to stimulate macrophages through different mechanisms. Complement receptor type 3 and scavenger receptor failed to mediate the cell activation induced by plant polysaccharides.

Assessment of certain neutrophil receptors, opsonophagocytosis and soluble intercellular adhesion molecule-1 (ICAM-1) following thermal injury

Polymorphonuclear leukocytes (PMLs) play a key role in host defense, and phagocyte dysfunction has been associated with increased susceptibility to infections in patients with thermal injury. Intercellular adhesion molecule-1 (ICAM-1) plays a role in leukocyte accumulation and extravasation. Therefore, the aim of the present study was to assess the PMLs expression of opsonin receptors: Fcγ RIII, CR1 and CR3; opsonophagocytosis of PMLs and plasma soluble ICAM-1. Flow cytometric analysis (FCM) was used to study PMLs expression of IgG Fc-receptor III (Fcγ RIII) as well as the complement receptors CR1 (receptor for C3b) and CR3 (receptor for C3bi) in 23 patients with large burns. Analysis of PML complement- and immunoglobulin-mediated phagocytosis of Candida albicans were performed in parallel using the phagocytic index. Plasma sICAM-1 was determined using ELISA. This study revealed a significant increase, with variable degrees, in CR1 and CR3-dependent fluorescence, complement-mediated phagocytosis of C. albicans and plasma sIGAM-1 that started at day 2 and remained for about 20 days before normalization. In contrast, Fcγ RIII-dependent fluorescence and Ig-mediated phagocytosis were significantly decreased versus the control values. These results demonstrate significant changes of PMLs opsonin receptors expression and opsonophagocytosis documenting systemic activator of PMLs after large burns. In addition, elevation of plasma sICAM-1 may enhance the harmful effect of neutrophil activation through leukocyte accumulation and extravasation through endothelial damage in skin and in lung.

The Effects of HIV Viral Load on the Phagocytic Activity of Monocytes Activated with Lipopolysacchae from Oral Microorganisms

A study was undertaken to determine whether viral load status in HIV' patients has any potential effect on monocyte phagocytic hnction both before and after challenge of the: monocytes with lipopolysaccharide (LPS) isolated from oral microorganisms. LPS of two putative periodontal pathogens Porphyromonas gingivalis (P. gingivalis;) and Fusobacterium nucleaturn (F. nucleatum) was prepared. Whole blood samples in EDTA were collected from 30 HIV± patients presenting for dental care at the University of Maryland. Control samples were prepared from appropriate uninfected individuals. Viral load was determined using quantitative RT-PCR (AmplicorR Roche Diagnostics). Phagocytic function was determined using FITC labeled Saccharomyces species in resting isolated monocytes and in cells after 24 h stimulation with 1 mg/ml of LPS of P. gingivlis or F. nucleatum. Immunohistochemical staining was performed for complement receptor CR-1 (CD-35) on phagocyte cells. In HIV± patients with high viral load (>10,000 copies/ml), 13.5% of isolated resting monocytes demonstrated phagocytic activity, while 23% of the resting control monocytes from non-infected individuals showed phagocytic function. When the monocytes were stimulated with 1 mg/ml of LPS of F. nucleatum phagocytic activity was observed in 18.5% of monocytes in patients with high viral load, 33.5% with moderate viral load (400-10,00 copies/ml) and 51% with low viral load (<400 copies/ml), while 62% of the control rnonocytes demonstrated phagocytic activity. Stimulation of monocytes with LPS of P. gingivalis showed similar results Complement receptor CD-35 showed a 50% decrease in expression in HIV± patients with high viral load. A progressive decrease in monocyte/macrophage phagocytic function and CD-35 expression with and without oral LPS activation occurs after HIV infection and this trend appears to be accentuated in patients with high viral load. This relationship may contribute to increased susceptibility to oral opportunistic infections in advanced HIV± patients.

Is complement a target for therapy in renal disease?

Complement deposition in the injured kidney is common, especially in glomerulonephritis. The precise role of the complement system in the mediation of tissue injury in the kidney has been defined in recent years, and this has assumed extra importance with the recent development of specific forms of therapy directed at the complement pathway. As well as the induction of cell lysis, complement has many subtle effects on cell biology, particularly on endothelial cells. Complement components are produced locally in the kidney. Detailed studies of certain rare forms of nephritis have provided evidence that complement activation can directly cause tissue injury. Appreciation of the importance of complement in hyperacute rejection of xenotransplants has given new impetus to the development of complement inhibitors. A narrative review is provided, with a brief overview of the complement pathway and its regulatory mechanisms, mechanisms of complement-induced tissue injury, local complement production, and the renal consequences of complement dysregulation. Currently available forms of therapy aimed at the complement system are reviewed, and possible future therapeutic strategies are suggested. The complement system plays a direct causal role in tissue injury in certain forms of renal disease. Specific forms of therapy are becoming available that can selectively interrupt complement activation or promote its regulation. Much of the drive for the development of these therapies comes from the field of xenotransplantation, but these forms of therapy should also be tested in various primary renal diseases.

Time course of microglia activation and apoptosis in various brain regions after permanent focal cerebral ischemia in mice.

We investigated the temporal course of microglia activation in different brain regions after permanent middle cerebral artery (MCA) occlusion in mice and compared this microglia response with the appearance of apoptotic cells, Microglia activation and morphological changes of microglial cells were visualized using an immunohistochemical method with a polyclonal antibody recognizing the mouse CR3 complement receptor. Cells showing morphological and biochemical features of apoptosis were identified using the terminal deoxynucleotidyl transferase nick end-labeling (TUNEL) method and light microscopy. As early as 30 min after onset of MCA occlusion activated microglia with hypertrophic cell bodies and stout processes were detected in the periphery of the ischemic lesion as identified by diffusion-weighted magnetic resonance imaging. A wider distribution and a progressive increase in the number of activated microglia was found with increasing time. Only few TUNEL-positive cells with apoptotic features were observed within the lesion area at 6 h after onset of cerebral ischemia. From 12 h after MCA occlusion onward a tremendous increase in the number of TUNEL-positive cells was found. Within the thalamus from 24 h onward microglia cells with few processes, irregular morphology and fragmented appearance were detected. Microglia activation in the thalamus progressed up to 4 weeks after MCA occlusion, but had declined after 90 days. Neuronal degeneration in the thalamus as determined by anti-neuronal nuclei immunohistochemistry progressed from 6 days after MCA occlusion onward. Only a few TUNEL-positive cells were found in the thalamus. In summary, microglia activation both in the primary cortical lesion area and in the secondarily affected thalamus preceded the manifestation of tissue injury. These observations encourage further studies on the role of microglia in focal cerebral ischemia.

Association of Immune Response and Neuronal Cell Death in the Rat Brain After Kainic Acid or Electrical Kindling Treatment

Purpose: In this study, we tried to clarify the possible involvement of immune responses in epilepsy. This attempt was based on our previous reports on the occurrence of immune responses in the rat hippocampus undergoing kainic acid (KA)‐induced neuronal death and in the sclerotic hippocampus of patients with temporal lobe epilepsy. However, it remains unclear whether these immune responses elicited neuronal degeneration in the sclerotic hippocampus. We investigated the relation between neuronal death and microglial activation in rats either pretreated with KA or submitted to electrical kindling. Methods: KA treatment: Adult male Sprague‐Dawley (SD) rats received a single intraperitoneal injection of KA (12 mg/kg). Only rats showing generalized limbic seizures were used in this study. Rats injected with physiological saline were used as controls. At 6, 12, and 18 h; 1, 2, 3, and 7 days; and 2, 4, 6, 8, 10, 12, and 16 weeks after the treatment, the animals were killed. Electrical kindling: Adult male SD rats were stereotaxically implanted with a bipolar electrode into the left perforant pathway near the angular bundle. Two weeks after electrode implantation, rats were submitted to daily kindling. The unrestrained awake rats received kindling stimulation with a 2‐s train of 60‐Hz biphasic constant‐current 1.0‐ms square‐wave pulses twice a day (6 days per week). The evoked behavioral seizures were classified according to standard criteria. Twenty‐four hours after each behavioral seizure stage, the rats were hlled. For rapid kindling, the unrestrained awake animals were subjected to electrical kindling with hourly and repeated electrical stimulation until generalized seizures were elicited 5 times. Sham‐operated rats served as controls. At 1, 3, 6, 9, and 15 days after the final stimulation, the animals were killed. The activated microglial cells and T lymphocytes were detected immunohistochemically with the avidin‐biotinylated peroxidase complex method. The primary antibodies were directed to complement receptors CR3 (OX‐42). MHC 1 (OX‐18), MHC II (OX‐6). and T‐cell surface antigens. Neuronal death was evaluated by determining DNA fragmentation. For this purpose, coronal cryostat sections of the brain were processed according to the TUNEL technique (in situ end‐labeling method). Results: In control rats, numerous resting microglial cells were distributed throughout the brain. However, neither MHC I‐ nor MHC II‐positive staining was observed in the brain. In KA‐treated rats, reactive microglial cells with hypertrophic bodies and thick processes appeared in the CA1 and CA3 fields of the hippocampus, hilus of the dentate gyms, thalamus, amygdala, and piriform cortex as early as 6 h after injection. The number of these reactive microglial cells quickly increased and reached the maximum at 7 days after the KA injection. The expression of MHC I on reactive microglia, DNA fragmentation, and appearance of cytotoxic/suppressor T lymphocytes were noted at 18 h after injection. The helper/inducer T lymphocytes amd MHC II–positive microglial cells were detected in the same brain regions at 48 h after injection. The fragmentation of DNA and T lymphocytes was detected until 4 and 8 weeks, respectively. After 16 weeks, MHC I‐ or MHC II–positive reactive microglial cells or both were reduced in number. In the daily‐kindling model, only a small number of reactive microglia was observed. In contrast, DNA fragmentation, MHC I‐ or MHC II–positive cells, and T lymphocytes were not detected. In the rapid‐kindling model, there were no activated microglial cells, DNA fragmentation, MHC I‐ amd MHC II–positive cells, or T lymphocytes. Conclusions: In the kindling model of epilepsy, neither DNA fragmentation nor immune responses were detected. The result indicates that epileptic seizures do not depend on the immune responses. In the KA‐treated model of epilepsy, immune responses were closely related to DNA fragmentation, suggesting an association of immune responses with neuronal death. We therefore suggest that immune responses play an important role in the neuronal death process induces by KA.

The complement system.

Work in the authors' laboratories is supported by grants from the Arthritis and Rheumatism Council and the Wellcome Trust.

Immunohistological studies on macrophages in lymph nodes of onchocerciasis patients after treatment with ivermectin.

The role of macrophages in the killing and elimination of microfilariae (mf) was studied immunohistologically in 14 lymph nodes from 10 patients with generalized onchocerciasis 20-68 h after treatment with a single oral dose of 150 microg/kg ivermectin. Mf with signs of damage at light microscopical level were surrounded by a cellular infiltrate comprising macrophages, eosinophils and neutrophils, whereas light microscopically intact mf mostly showed no cellular reaction. Resident mature macrophages expressing the CD 68 epitope usually neither migrated nor attached to damaged mf, especially on the first and second day after ivermectin treatment. However, many young invading macrophages labelled for the L1 protein (antibodies 27 E 10, MAC 387, S 36.48 and 8.5C2) were found within the cellular infiltrate around damaged mf and in adherence to the mf in all lymph nodes after ivermectin treatment. Free L1 protein was observed on the cuticle of the mf. The attacking macrophages contained increased amounts of the enzymes lysozyme, alpha-1-antichymotrypsin and alpha-1-antitrypsin compared to resident macrophages. Free enzymes were found on the cuticle of the mf and around them, indicating a role of these enzymes in the inflammatory reaction to the parasites. The attacking macrophages were strongly labelled for human HLA-DR and they showed further an increased expression of the complement receptors CR1 (CD 35) for C3b and CR3 (CD 11b) for C3 bi in comparison to resident macrophages and thus were considered as activated macrophages. Rarely fragments of mf were seen within multinuclear macrophages. We conclude that young activated macrophages play a major role in the elimination of mf transported to the regional lymph nodes after ivermectin treatment. The immunohistological findings are in accordance with the assumption that these activated macrophages together with granulocytes contribute to the killing of the damaged mf. They also help to limit the damage of the host tissue by release of alpha-1-antichymotrypsin and alpha-1-antitrypsin.

Integrin CR3 mediates the binding of nonspecifically opsonized Borrelia burgdorferi to human phagocytes and mammalian cells.

Like other pathogens, the spirochete Borrelia burgdorferi, the agent of Lyme disease, possesses multiple pathways for cell binding; adhesion to phagocytic cells is of particular interest since it reportedly occurs even in the absence of specific antibodies. This study sets out to investigate how B. burgdorferi binds to human polymorphonuclear leukocytes (PMNs) when an exogenous complement is added and how the CR3 complement receptor, known as Mac-1 or alpha(m)beta2 integrin, is involved in the binding process. Experiments performed on PMNs and CHO Mac-1-expressing cells demonstrate that binding is inhibited by monoclonal anti-iC3b site antibodies, fibrinogen, and N-acetyl-D-glucosamine. These findings, which are not present with non-Mac-transfected CHO cells, indicate that the integrin alpha(m)beta2 acts as a receptor for spirochetes in nonimmune phagocytosis; furthermore, binding occurs on different domains of the CD11b subunit, involving the iC3b site and the lectin domain. The interaction of B. burgdorferi with alpha(m)beta2 integrin adds a novel pathway to Borrelia-phagocyte binding; not only does this binding affect the early stages of phagocytosis, but also it can influence the effector intracellular mechanisms which are activated by the beta2 integrin, as are the cytotoxic mechanisms.

Identification of a Gal/GalNAc lectin in the protozoan Hartmannella vermiformis as a potential receptor for attachment and invasion by the Legionnaires' disease bacterium.

The Legionnaire's disease bacterium, Legionella pneumophila, is a facultative intracellular pathogen which invades and replicates within two evolutionarily distant hosts, free-living protozoa and mammalian cells. Invasion and intracellular replication within protozoa are thought to be major factors in the transmission of Legionnaire's disease. Although attachment and invasion of human macrophages by L. pneumophila is mediated in part by the complement receptors CR1 and CR3, the protozoan receptor involved in bacterial attachment and invasion has not been identified. To define the molecular events involved in invasion of protozoa by L. pneumophila, we examined the role of protein tyrosine phosphorylation of the protozoan host Hartmannella vermiformis upon attachment and invasion by L. pneumophila. Bacterial attachment and invasion were associated with a time-dependent tyrosine dephosphorylation of multiple host cell proteins. This host cell response was highly specific for live L. pneumophila, required contact with viable bacteria, and was completely reversible following washing off the bacteria from the host cell surface. Tyrosine dephosphorylation of host proteins was blocked by a tyrosine phosphatase inhibitor but not by tyrosine kinase inhibitors. One of the tyrosine dephosphorylated proteins was identified as the 170-kD galactose/N-acetylgalactosamine-inhibitable lectin (Gal/GalNAc) using immunoprecipitation and immunoblotting by antibodies generated against the Gal/GalNAc lectin of the protozoan Entamoeba histolytica. This Gal/GalNAc-inhibitable lectin has been shown previously to mediate adherence of E. histolytica to mammalian epithelial cells. Uptake of L. pneumophila by H. vermiformis was specifically inhibited by two monovalent sugars, Gal and GalNAc, and by mABs generated against the 170-kD lectin of E. histolytica. Interestingly, inhibition of invasion by Gal and GalNAc was associated with inhibition of bacterial-induced tyrosine dephosphorylation of H. vermiformis proteins. High stringency DNA hybridization confirmed the presence of the 170-kD lectin gene in H. vermiformis. We conclude that attachment of L. pneumophila to the H. vermiformis 170-kD lectin is required for invasion and is associated with tyrosine dephosphorylation of the Gal lectin and other host proteins. This is the first demonstration of a potential receptor used by L. pneumophila to invade protozoa.

Genetic influences on cellular reactions to brain injury: Activation of microglia in denervated neuropil in mice carrying a mutation (WldS) that causes delayed Wallerian degeneration

This study examines the relationship between the appearance of degenerative changes in synaptic terminals and axons and the activation of microglia in denervated neuropil regions of normal mice of the C57BL/6 strain and mutant mice (Wld(S)), in which Wallerian degeneration is substantially delayed. The time course of degenerative changes in synaptic terminals and axons was assessed using selective silver staining. Microglial cells were identified by immunostaining for Mac-1, a monoclonal antibody to the CR3 complement receptor, and by histochemical staining for nucleoside diphosphatase (NDPase). Increased argyrophilia, indicative of degenerative changes, was evident as early as 1 day postlesion in normal mice, but was not seen until 6-8 days in mice with the Wld(S) mutation. Microglial activation in normal C57BL/6 mice was evident by 24 hours postlesion, as evidenced by increased immunostaining for Mac-1, increased histochemical staining for NDPase, and morphological changes indicative of an activated phenotype (short, thick processes). Quantitative evaluation of immunostaining for Mac-1 revealed that peak activation occurred between 2 and 6 days postlesion with a return to a quiescent phenotype by 12 days. In contrast, the microglial response was significantly delayed and prolonged in mice bearing the Wld(S) mutation. Activated microglia were not seen within the deafferented area until 6 to 8 days postlesion and peak activation occurred between 12 and 20 days postlesion. These data suggest that the response of microglia in denervated neuropil zones is triggered by the same types of degenerative changes that cause increased argyrophilia as detected by selective silver staining methods.

Effect of sublethal X-irradiation on follicular trapping of immune complexes in the mouse lymph node.

In one set of experiments, the effect of sublethal X-irradiation on the 24 h localization of subcutaneously injected immune complexes in the lymph node follicles was studied in mice which were given HRP-anti-HRP complexes into the rear footpad at 1-3 weeks after irradiation and killed 1 day later. The 24 h follicular localization of injected immune complexes in draining popliteal nodes was severely impaired at 7 days after irradiation, at which time residual follicles were markedly depleted of B lymphocytes. In following weeks, residual follicles began to be repopulated, and the 24 h follicular localization of immune complexes became restored. Follicular dendritic cells (FDC), as was detected by the in vitro trapping assay and/or by the immunostaining for complement receptors CR1, were present in lymph nodes at any time after irradiation. Another group of mice were given HRP-anti-HRP complexes at 6 days of X-irradiation and killed from 15 min to 24 h later. Following the injection, complexes localized in residual follicles in draining nodes within 15 min but soon diminished in density and finally disappeared by 24 h after injection. It is obvious that sublethal irradiation affect neither transport of immune complexes to lymph node follicles nor their localization in these follicles. Rather rapid disappearance after temporal localization of immune complexes in residual follicles irradiated mice indicates that persisting FDC were unable effectively to trap immune complexes which were transported and localized in residual follicles. Ineffective trapping by FDC of immune complexes temporally localized in residual follicles is discussed in relation to the depletion of follicular B lymphocytes due to X-irradiation.