Molecular mechanism of monocyte predominant infiltration in chronic inflammation: mediation by a novel monocyte chemotactic factor, S19 ribosomal protein dimer

A novel monocyte chemotactic factor, a cross-linked homodimer of S19 ribosomal protein (RP S19) was initially isolated from a rheumatoid arthritis synovial lesion. The RP S19 dimer causes the monocyte specific chemotaxis in vitro and the monocyte predominant infiltration in vivo, via its agonistic and antagonistic effects on the C5a receptors of monocytes and polymorphonuclear leukocytes, respectively. The agonistic effect is attributed to the similarity of regional structures between RP S19 and C5a, the complement C5-derived leukocyte chemotactic factor, although overall homology of the amino acid sequence between these molecules is only 4%. The antagonistic effect depends upon the C-terminal portion of RP S19. The RP S19 dimer is produced and released by apoptotic cells, and this dimer recruits monocytes from the circulation to the apoptotic lesion. The infiltrated monocytes/macrophages engulf the apoptotic cells, translocate to regional lymph nodes via lymphatics and present the antigenic information of the apoptotic cells to the T cell repertoire. In this manner, the apoptotic cell clearance system connects to the acquired immune system. The innate and acquired immune mechanisms, mediated by the RP S19 dimer, participate in the pathology of inveterate chronic inflammation such as rheumatoid arthritis.     

Role of ribosomal protein S19-like plasma protein in blood coagulum resorption

Western blot analyses and monocyte chemoattraction analyses of guinea pig plasma and serum indicated the presence of a plasma protein indistinguishable from ribosomal protein S19 and the cross-linked dimerization of it gaining monocyte chemotactic capacity in association with blood coagulation as in the case of human. When coagula preformed in vitro were intraperitoneally inserted into guinea pigs, they were rapidly covered by macrophages within 24 h concomitant with an intra-coagulum macrophage infiltration. Differences were observed between the surface macrophages and the penetrating macrophages in ultrastructural, histochemical and immunohistochemical analyses. The inserted coagula were resorbed by day 7. When either anti-RP S19 antibodies or Gln137Asn-RP S19, a competitive inhibitor against RP S19, was premixed into the inserted coagulum, the attachment and penetration by macrophages decreased and the coagulum resorption retarded. These results indicate the role of the plasma RP S19-like molecule in coagulum resorption via macrophage recruitment.     

A novel monocyte chemotactic factor that connects innate immunity and acquired immunity

A monocyte chemotactic factor was separated from rheumatoid arthritis synovium, and identified as a homo-dimer of S19 ribosomal protein. When S19 protein was treated with the plasma transglutaminase, an inter-molecular isopeptide bond was formed between Lys122 and Gln137, and the chemotactic activity appeared. The S19 protein dimer caused chemotaxis via the receptor on monocytes to C5a, the complement C5-derived chemotactic factor. This dimer antagonized the C5a receptor on neutrophils. This dimer was released from apoptotic cells, and functioned in the phagocytic clearance of these cells by recruiting circulating monocytes. After engulfment, the macrophages moved to regional lymph nodes, and presented apoptotic cell-derived antigens to T cells. T cells proliferated and activated B cells, and eventually the IgM antibody response was observed. Cooperation between the innate immune response and the acquired response would induce an effective host defense primarily against viral infection.     

Persistence of Adenovirus 5 in Guinea Pigs

One intracardiac inoculation of adenovirus 5 in guinea pigs leads to virus persistence in different organs, viz., 5 days in lungs and liver, 14 days in blood and lymph nodes, and 56 days or more in the spleen. After cultivation of tissue cells for 1 week, virus was recovered from blood, lymph nodes, or spleen lymphocytes, but virus could be detected directly in cells only when organs were removed within 48 h of inoculation. To determine how the virus persisted in low concentrations and as a latent infection, spleens were primarily selected for study by three techniques: homogenization of spleens, suspended Maitland fragment cultures, and in vitro cultivation of spleen cells. The last procedure showed virus in fibroblast-like cells (probably macrophages or reticuloendothelial cells) for 56 days after infection of guinea pigs. With other methods, the virus was found only within the first 2 days after inoculation.     

Cytomegalovirus-induced mononucleosis in guinea pigs.

The effects of cytomegalovirus (CMV) infection on hematopoietic and lymphoid tissues were studied in guinea pigs. Blood parameters, histopathology, and virus distribution in the bone marrow, spleen, lymph nodes, and thymus were assessed during primary nonlethal acute and chronic guinea pig CMV infection. Transient hematological changes comparable to those seen in human CMV mononucleosis were observed during acute infection. These included anemia and leukocytosis with atypical lymphocytes. Splenomegaly and stimulation of spleen and lymph node T- and B-cell areas were also noted. These changes occurred at the peak of virus recovery from all tissues tested, as well as from macrophages and B- and T- cell-enriched spleen subpopulations. Virus was cleared rapidly from blood and bone marrow; blood counts, spleen size, and histology returned to normal within 1 month after virus inoculation. However, guinea pigs failed to eliminate the virus completely from lymphoid tissues, since virus persisted in splenic macrophage and B-lymphocyte-enriched populations during chronic infection. The data suggest that CMV-infected mononuclear cells play a role in the establishment of generalized acute infection and virus persistence.     

Immunohistochemical study of lymphoid tissues in adjuvant arthritis (AA) by image analysis; relationship with synovial lesions

The aim of this study was to examine leucocyte populations in lymphoid organs during AA and to ascertain the relationship with lesions in synovial joints. Popliteal lymph nodes, spleen and knee synovial membranes were removed from both healthy and AA rats at intervals of 3-4 days over a 3-week period. Cryostat sections were stained with MoAbs directed against lymphocyte and macrophage subpopulations, and studied by image analysis. Throughout the arthritic period, high numbers of ED1+ and ED3+ macrophages were seen in both lymphoid compartments and intercellular adhesion molecule-1 (ICAM-1) expression also increased in some zones of lymph nodes and spleen. The percentages of CD4+ and CD8+ cells rose in the splenic zones studied but fell in the lymph node cortex. Very few natural killer (NK) cells were found in lymphoid tissues, but the number rose after AA induction. In synovia from AA rats, ED2+ macrophages proliferated but alpha/beta T cell infiltration was only occasionally observed, accompanied by ED1+ cells and ICAM-1 expression. In conclusion, synovitis developing after AA induction seems to be caused directly by macrophages and indirectly by lymphocytes placed both in popliteal lymph nodes and spleen.     

Macrophages and the activity of high endothelial venules. The effect of interferon-gamma

Interruption of the afferent lymphatic vessels of rat popliteal lymph nodes led to the disappearance of the monoclonal antibody ED3-reactive subcapsular sinus macrophages within 3 weeks, but had no effect on the ED1+ macrophages in the paracortical area. This disconnection of the afferent lymph flow to the popliteal lymph node also reduced the capacity of high endothelial venules (HEV) to bind lymphocytes and led to a flattening of the HEV. Activating factors or cells in the incoming lymph might be responsible for the maintenance and function of several different cell populations and we therefore wished to determine if the effects of interruption could be restored by injection of recombinant rat interferon-gamma (rIFN-gamma). Injection of rIFN-gamma directly into operated lymph nodes could mediate an apparent increase of ED1+ cells within 24 h but rIFN-gamma could not restore the macrophage subpopulation in the subcapsular sinus, as recognized by monoclonal antibody ED3. Restoration of the decreased binding capacity of HEV could not be observed with the doses and time points tested, suggesting that HEV are a distinct type of endothelium.     

Recognition of apoptotic cells by human macrophages: inhibition by a monocyte/macrophage-specific monoclonal antibody

Cells undergoing death by apoptosis are rapidly engulfed by phagocytes in vivo, a highly efficient process which prevents leakage of potentially dangerous intracellular contents from dying cells to neighboring tissue. We have tested a panel of monoclonal antibodies (mAb) specifying a range of human monocyte/macrophage surface antigens for their capacity to inhibit the in vitro recognition of apoptotic cells by human peripheral blood monocyte-derived macrophages. The results identify the antigen defined by the 61D3 mAb, a widely-used marker of monocyte/macrophage lineage cells, as an important mediator of apoptotic cell recognition. In our system, apoptotic, but not viable, cells were recognized by the cultured macrophages and 61D3 was found to inhibit the recognition of all apoptotic cell types tested, including Ca²⁺ ionophore-treated or growth factor-depleted B and T lymphocyte lines, tonsillar germinal center B cells, irradiated peripheral blood lymphocytes and senescing neutrophils. Furthermore, the apoptotic cell recognition pathway specified by 61D3 could be distinguished from that involving the macrophage α_vβ₃ vitronectin receptor which has been shown previously to play an important role in the recognition of apoptotic cells. These results provide further evidence that the mechanisms underlying rapid clearance of apoptotic cells involve multiple phagocyte receptors.     

The dendritic cells of the guinea pig popliteal lymph node: identification and classification of cells observed by scanning electron microscopy.

Scanning electrom microscopy of guinea pig popliteal lymph nodes revealed that 3 types of cells adhered to glass and possessed extensive cytoplasmic processes. Two of these types were considered to represent extremes of a macrophage population on the basis of possession of surface receptors for cytophilic Abs, complement and immunoglobulin, as well as their distribution, quantity and morphology. The third cell type was characterized by possession of large numbers of filamentous dendritic processes. On the basis of morphology, quantity, distribution and the absence of receptors, this cell was considered to represent a cell type clearly distinct from the other cells observed in the normal lymph node.     

Natural IgM and innate immune collectin SP-D bind to late apoptotic cells and enhance their clearance by alveolar macrophages in vivo

Innate immune collectin surfactant protein D (SP-D) and natural immunoglobulin M (IgM) are two soluble proteins. These opsonic proteins are good candidates for enhancing late apoptotic cell clearance. However, effects of these proteins on late apoptotic cell clearance in the lungs are not clearly established. We have recently shown that SP-D can bind several immunoglobulin isotypes, including IgM. Here we hypothesized that IgM and SP-D bind to late apoptotic cells and enhance their clearance from the lungs. We show that IgM and SP-D bind to late apoptotic secondary necrotic cells, and that IgM and SP-D either co-localize to the same regions or to different regions of late apoptotic Jurkat T cells. Mouse alveolar macrophages internalized late apoptotic cells, in vivo. We induced lung inflammation in mice using LPS and show that airway IgM and SP-D levels and the clearance of late apoptotic cells by alveolar macrophages increases under these conditions. We then coated late apoptotic cells with IgM, SP-D, or both and instilled them into the mouse airways. We found that alveolar macrophages internalize IgM- and SP-D-coated late apoptotic cells more effectively than uncoated late apoptotic cells, in vivo. None of these conditions cause inflammation in the na?ve lungs. Therefore, these data suggest that both IgM and SP-D effectively opsonize late apoptotic cells and directly enhance their clearance by alveolar macrophages in the lungs.     

Establishment of cutaneous Leishmania enriettii infection in hamsters.

A model of a self-healing type of cutaneous leishmaniasis was established in hamsters using the guinea pig parasite Leishmania enriettii. L. enriettii was passaged several times in hamsters without losing its infectivity for guinea pigs or for hamsters. The course of the infection in hamsters resembled that of guinea pigs, with the exception that the lesion at the site of parasite inoculation did not ulcerate and no metastatic lesions developed spontaneously. Moreover, unlike guinea pigs, infected or recovered hamsters were skin test unresponsive to various preparations of L. enriettii antigens. However, histological examination of draining lymph nodes showed features of a cell-mediated immune response, and in vitro inhibition of macrophage migration was demonstrable using peritoneal exudate cells from recovered animals and specific leishmanial antigen. Antibody was demonstrable by indirect immunofluorescence starting 1 week after infection. Recovered animals were immune to reinfection; however, the passive transfer of peritoneal exudate cells or serum from recovered animals did not confer protection against L. enriettii infection in normal animals.     

Pathology and viral antigen distribution following experimental infection of sheep and goats with capripoxvirus

Current understanding of capripoxvirus pathogenesis is limited since there have been no detailed studies examining cell tropism at well-defined intervals following infection. We undertook time-course studies in sheep and goats following inoculation of sheeppox or goatpox viruses in their respective homologous hosts, and examined tissues by light microscopy. A monoclonal antibody generated to a sheeppox virus core protein was used for immunohistochemical detection of viral antigen in tissue sections. Lesions and virus antigen were observed consistently in the skin, lung and lymph nodes. Antigen was detected at 6 and 8 days post inoculation for skin and lung, respectively, within cells which appeared to be of monocyte/macrophage lineage. In sheep skin capripoxvirus immunoreactivity was detected within previously unreported large multinucleated cells. In the lung, double immunolabelling detected the simultaneous expression of capripoxvirus antigen and cytokeratin indicating the presence of virus within pneumocytes. Lung double immunolabelling also detected the expression of capripoxvirus antigen in CD68(+) cells, confirming the presence of viral antigen within macrophages. Based on early detection of infected macrophages, dissemination of virus within the host and localization to tissues likely occurred through cells of the monocyte/macrophage lineage. Histological findings revealed similarities with both monkeypox and smallpox, thus capripoxvirus infection in sheep and goats may represent useful models with which to study strategies for poxvirus-specific virus vaccine concepts and therapeutics.     

Transformation of guinea pig leukocytes by coinfection with human T-cell lymphotropic virus types I and II

OBJECTIVE: The susceptibility of guinea pigs to human T-cell lymphotropic virus (HTLV) infection and of their cardiac blood mononuclear cells (CBMCs) to HTLV-induced transformation were investigated. STUDY DESIGN/METHODS: Guinea pig CBMCs were cocultured with HTLV-infected cell lines. Guinea pigs were then inoculated with transformed guinea pig CBMCs. RESULTS: The coculture experiment gave rise to a guinea pig cell line, GP-1, that was coinfected with both HTLV-I and HTLV-II as shown by immunofluorescence staining, electron microscopy, polymerase chain reaction (PCR) using primers specific for the pol region of each virus, and Southern blot hybridization. The GP-1 cell line expressed T-cell markers and monocyte/macrophage markers. Three guinea pigs given an intraperitoneal inoculation of GP-1 cells seroconverted for HTLV-I and became positive for HTLV-I, HTLV-II, or both, as confirmed by PCR. CONCLUSIONS: Guinea pigs and their CBMCs can be infected with HTLV-I and HTLV-II. This animal system may be useful as an experimental model of HTLV-I and HTLV-II infection.     

Binding of Candida albicans yeast cells to mouse popliteal lymph node tissue is mediated by macrophages.

We previously reported that Candida albicans yeast cells adhere to the macrophage-rich medullary and subcapsular sinus areas of mouse lymph node tissue. To determine whether the yeast cell-lymph node interaction is mediated by macrophages, the effect of specific elimination of macrophages on yeast cell binding was studied, and yeast cell adherence was correlated with the ingestion of India ink by lymph node cells. Macrophage elimination was done by use of liposome-containing dichloromethylene diphosphonate (L-Cl2MDP). Mice were injected in the hind footpads with the L-Cl2MDP preparation, popliteal lymph nodes were removed 5 days later, and yeast cell adherence was determined by an ex vivo binding assay. As controls, lymph nodes from mice that received footpad injections of either phosphate-buffered saline (PBS) alone or liposome-containing PBS were used. Use of macrophage- and neutrophil-specific monoclonal antibodies in tissue immunostaining showed that the L-Cl2MDP treatment eliminated macrophages but not neutrophils from the medullary and subcapsular sinus areas of the popliteal lymph nodes. A striking reduction of yeast cell adherence occurred with lymph nodes from L-Cl2MDP-treated mice compared with lymph nodes from control animals. The lymph node-yeast cell binding patterns of L-Cl2MDP-treated and control mice were the same regardless of mouse strain, sex, or T-cell competency. Results of India ink experiments, in which India ink was injected into footpads of mice and was rapidly taken up by popliteal lymph node macrophages, showed a strong correlation between yeast adherence and India ink staining of cells. In addition, the interaction of yeast cells with lymph node tissue from normal mice was not significantly affected by the addition of two extracellular matrix proteins, fibronectin and laminin, during the ex vivo adherence assay. These data indicate that medullary and subcapsular sinus lymph node macrophages express an adhesion system similar to that described for mouse splenic marginal zone macrophages.     

Pathological and virological features of arenavirus disease in guinea pigs. Comparison of two Pichinde virus strains.

A guinea pig passage-adapted strain of the arena-virus Pichinde (adPIC) is highly virulent in inbred guinea pigs, whereas the related strain PIC3739 is attenuated. Both viruses were macrophage tropic and infected peritoneal, splenic, liver, and alveolar macrophages during experimental Pichinde virus infection. Infection with the virulent strain was associated with unlimited viral replication in the face of exaggerated delayed-type hypersensitivity response, manifested by the macrophage disappearance reaction. Histopathological lesions unique to adPIC-infected guinea pigs included intestinal villus blunting with mucosal infiltration by pyknotic debris-laden macrophages and apoptosis of crypt epithelial cells. Splenic red pulp necrosis was also significantly associated with adPIC infection but not PIC3739 infection. These findings may provide clues to the pathogenesis of a group of poorly understood human viral hemorrhagic fevers.     

Melanosis coli. A consequence of anthraquinone-induced apoptosis of colonic epithelial cells.

A condition closely resembling human melanosis coli was induced in the guinea pig large intestine by daily oral administration of the anthraquinone danthron. Each treatment caused a transient, dose-related wave of apoptosis of the colonic surface epithelial cells. Most of the resulting apoptotic bodies were phagocytosed by intraepithelial macrophages and carried by them through fenestrae in the epithelial basement membrane to the lamina propria. Here, the apoptotic bodies were transformed into typical lipofuscin pigment in macrophage heterolysosomes. Continued danthron administration caused progressive accumulation of pigmented macrophages in the bowel wall, whereas ongoing migration of pigmented macrophages to regional lymph nodes resulted, after danthron was ceased, in sequential loss of the pigmented cells from the superficial and deep lamina propria. Examination of colonic biopsies from patients with melanosis coli shows increased numbers of apoptotic bodies in the surface epithelium and lamina propria, suggesting implication of the same cellular processes in the formation of the pigment in man.     

IGM is required for efficient complement mediated phagocytosis of apoptotic cells in vivo

A variety of complement components have been detected on apoptotic cells and proposed to facilitate recognition and/or ingestion by phagocytes. The triggers for complement activation remain uncertain. To determine the role of IgM in classical pathway activation and clearance of apoptotic cells in vitro and in vivo, we quantified these parameters in mice deficient in serum IgM (sIgM). Phagocytosis by bone marrow-derived macrophages of apoptotic cells incubated with serum deficient in sIgM was markedly reduced, similar to apoptotic cells incubated with C1q deficient serum in vitro. Similarly, intraperitoneal clearance of apoptotic cells and cellular C3 deposition were significantly reduced in mice deficient in sIgM compared to wild-type mice. Clearance and C3 deposition were reconstituted by addback of IgM. In mice deficient in both sIgM and Clq, addback of both serum factors was required for restoration of clearance. These findings indicate that, on a quantitative basis, sIgM is a potent factor required for intraperitoneal phagocytosis of apoptotic cells, and further demonstrate that IgM and C1q work in concert to activate complement, resulting in C3 deposition on the apoptotic cell surface and ultimately, efficient clearance of the apoptotic cell by macrophages.     

Distribution of a Korean strain of porcine reproductive and respiratory syndrome virus in experimentally infected pigs, as demonstrated immunohistochemically and by in-situ hybridization

In an experiment with 40 specific pathogen-free pigs aged 3 days, the distribution of a Korean isolate of porcine reproductive and respiratory syndrome virus (PRRSV) was assessed immunohistochemically and by in-situ hybridization for a period of 28 days after intranasal inoculation. The most consistent and intense labelling for PRRSV was in the lung, the virus persisting in pulmonary macrophages for at least 28 days. The middle lobe of the lung was the optimum site for the detection of PRRSV antigens and nucleic acids, and the interstitial macrophage was the main cell type in which PRRSV was identified. Other tissues and cells in which the virus was detected included macrophages and dendritic cells in the tonsil, lymph nodes, spleen and Peyer's patches, and macrophages in the hepatic sinusoids and adrenal gland. The experiment suggested that the pathogenesis of PRRSV infection may be summarized thus: initial entry of virus through tonsillar and pulmonary macrophages, followed within 3 days by viraemia and subsequent interstitial pneumonia.     

IGM is required for efficient complement mediated phagocytosis of apoptotic cells in vivo

A variety of complement components have been detected on apoptotic cells and proposed to facilitate recognition and/or ingestion by phagocytes. The triggers for complement activation remain uncertain. To determine the role of IgM in classical pathway activation and clearance of apoptotic cells in vitro and in vivo, we quantified these parameters in mice deficient in serum IgM (sIgM). Phagocytosis by bone marrow-derived macrophages of apoptotic cells incubated with serum deficient in sIgM was markedly reduced, similar to apoptotic cells incubated with C1q deficient serum in vitro. Similarly, intraperitoneal clearance of apoptotic cells and cellular C3 deposition were significantly reduced in mice deficient in sIgM compared to wild-type mice. Clearance and C3 deposition were reconstituted by addback of IgM. In mice deficient in both sIgM and C1q, addback of both serum factors was required for restoration of clearance. These findings indicate that, on a quantitative basis, sIgM is a potent factor required for intraperitoneal phagocytosis of apoptotic cells, and further demonstrate that IgM and C1q work in concert to activate complement, resulting in C3 deposition on the apoptotic cell surface and ultimately, efficient clearance of the apoptotic cell by macrophages.