Antimacrophage Serum Research Articles

Galectin-3 Expression and Secretion Links Macrophages to the Promotion of Renal Fibrosis

Macrophages have been proposed as a key cell type in the pathogenesis of renal fibrosis; however, the mechanism by which macrophages drive fibrosis is still unclear. We show that expression of galectin-3, a beta-galactoside-binding lectin, is up-regulated in a mouse model of progressive renal fibrosis (unilateral ureteric obstruction, UUO), and absence of galectin-3 protects against renal myofibroblast accumulation/activation and fibrosis. Furthermore, specific depletion of macrophages using CD11b-DTR mice reduces fibrosis severity after UUO demonstrating that macrophages are key cells in the pathogenesis of renal fibrosis. Disruption of the galectin-3 gene does not affect macrophage recruitment after UUO, or macrophage proinflammatory cytokine profiles in response to interferon-gamma/lipopolysaccharide. In addition, absence of galectin-3 does not affect transforming growth factor-beta expression or Smad 2/3 phosphorylation in obstructed kidneys. Adoptive transfer of wild-type but not galectin-3(-/-) macrophages did, however, restore the fibrotic phenotype in galectin-3(-/-) mice. Cross-over experiments using wild-type and galectin-3(-/-) macrophage supernatants and renal fibroblasts confirmed that secretion of galectin-3 by macrophages is critical in the activation of renal fibroblasts to a profibrotic phenotype. Therefore, we demonstrate for the first time that galectin-3 expression and secretion by macrophages is a major mechanism linking macrophages to the promotion of renal fibrosis.

Inflammation in surgical wound healing: Friend or foe?

In 1922, Dr Alexis Carrel1 Carrel A. Growth-promoting function of leucocytes. J Exp Med. 1922; 36: 385-391 Crossref PubMed Scopus (57) Google Scholar demonstrated that leukocytes were capable of producing factors that stimulated cellular proliferation. Since that time, the appropriate function of inflammatory cells has been generally considered indispensable for successful wound healing. Landmark studies in the early 1970s and 1980s demonstrated that immune cells, particularly macrophages, are critical to wound healing, and the role of macrophages as mediators of angiogenesis and fibroplasia has been established firmly. 2 Leibovich S.J. Ross R. The role of the macrophage in wound repair: a study with hydrocortisone and antimacrophage serum. Am J Pathol. 1975; 78: 71-100 PubMed Google Scholar , 3 Hunt T.K. Knighton D.R. Thakral K.K. et al. Studies on inflammation and wound healing: angiogenesis and collagen synthesis stimulated in vivo by resident and activated wound macrophages. Surgery. 1984; 96: 48-54 PubMed Google Scholar The study of wound inflammation continues to receive intense attention, with more than 1000 articles on the topic published within the past five years. There is little argument that proper leukocyte activity assists in microbial decontamination of wounds. In addition, there are several logical arguments in support of a role for leukocytes in healing, even for sterile wounds. First, phagocytic leukocytes, as well as lymphocytes, produce multiple growth factors that promote the repair process. Second, the cellular death and tissue remodeling that occur during injury and repair would seem to require phagocytic clearance for complete resolution. Finally, studies in a variety of model systems suggest that key inflammatory cytokines are critical to wound healing. 4 Gillitzer R. Goebeler M. Chemokines in cutaneous wound healing. J Leukoc Biol. 2001; 69: 513-521 PubMed Google Scholar Despite these previous findings, other recent studies challenge the established paradigm and suggest that the role of leukocytes in the repair of sterile or minimally contaminated wounds requires reevaluation.

Role of adherent spleen cells in the induction of cytotoxic activity by Toxoplasma lysate antigen.

In order to identify mechanisms responsible for the anti-tumor effects of Toxoplasma lysate antigen (TLA), we used an in vitro 51Cr release assay to study the functional properties of plastic-adherent cells during induction of splenic cytotoxic activity by TLA. Cytotoxic activity of non-adherent cells was measured in all experiments after a 6 days incubation. Induction of cytotoxic non-adherent cells by TLA required the presence of plastic-adherent spleen cells. In contrast, rhIL-2 alone was able to induce transformation of cytotoxic non-adherent cells from non-adherent spleen cells. Contact between adherent and non-adherent spleen cells was necessary for successful induction of cytotoxic non-adherent cells by TLA. Treatment of spleen cells with anti-macrophage serum prevented induction of cytotoxic activity by TLA. Biologically active IL-2 was not detected in culture supernatants of spleen cells exposed to TLA. These findings suggest that contact between TLA-sensitized non-adherent cells and macrophages is necessary for induction of cytotoxic cells in the presence of TLA. This contact, however, is not necessary for generation of IL-2-induced killer cells.

Characterization of cells comprising murine thymocyte multicellular complexes.

Characterization of complexes between thymocytes and thymic stromal cells was facilitated in the present study by demonstrating that complexes would reform if cells were incubated for 1.5 to 2 h in vitro at ambient temperature. Several immunologic approaches were used to determine the phenotype of complexed cells. Bound T cells were 97% double-positive (CD4 and CD8), 3% double-negative and greater than 99% CD3 positive by using immunoperoxidase immunohistology on cytospins. Five percent expressed the TCR beta-chain and 1 to 2% were IL-2R positive. The percentages were the same whether complexes were preformed in vivo or formed in vitro. Despite the apparent absence of single positive cells in complexes, when isolated CD4 or CD8 positive cells were tested for their ability to bind to adherent thymic macrophages, each subpopulation contained some cells which were capable of complex formation. When thymocytes were fractionated by density, steroid sensitivity or peanut agglutinin positivity then allowed to form complexes, cells with an immature phenotype had a greater propensity for complex formation. Central stromal cells all were class II MHC gene product (I-A and I-E) positive, expressed macrophage-associated Ag (B23.1 and MAC-1), were negative for cytokeratin but positive for vimentin, were reactive with a polyclonal antimacrophage serum, but did not express dendritic cell Ag (33D1). The data demonstrate that immature thymocytes bind exclusively to class II MHC gene product positive thymic macrophages. This binding step may play a role in the acquisition of T cell function in the thymus.

Die verhinderung der lymphohämatogenen Streuung als pathogenetische voraussetzung für die neurale persistenz des Herpes simplex Virus

In the experimental model of genital herpes simplex virus (HSV) infection of the mouse--in spite of abundant virus replication on the mucous membranes--no infectious virus can be isolated from the inflamed and swollen draining lymph nodes (DLN), contrary to the positive results in the lumbosacral nerves and their associated ganglia. Attempts to reactivate an abortive infection possibly established in lymph node or spleen cells by stimulation with phytohemagglutinin or lipopolysaccharide rendered no positive results, not even when adult (i.e. at least 6-week old), but immunodeficient mice were used as test animals. On the other hand, isolation of infectious virus from lymph node and--at a lesser rate--from spleen cells was successful in immature 4 to 6-week old mice, particularly when these had undergone pretreatment with cyclophosphamide, silica, antimacrophage serum and/or cortisone; 5 days post infectionem being the date of optimum virus yield. HSV-1 infected mice were more frequently positive than those with HSV-2, and genetically sensitive animals more so than resistant mice. The data indicate that the lymphohaematogenous spread of the virus is inhibited by means of an active defence mechanism, and that unspecific defence factors on the cellular level, probably macrophages and NK-cells, are essentially responsible. This reveals that the lethal generalized infection is prevented and the neural spread can gain its essential role in the pathogenesis of the HSV-infection.

Effect of macrophage depletion on growth and neovascularization of hamster buccal pouch carcinomas

The effect of macrophage depletion on growth and neovascularization of 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch carcinomas (HBPC) was evaluated by quantitating tritiated thymidine (3[H]TdR) incorporation by tumor cells and microvascular endothelium in light microscopic autoradiographs. Tumors that were depleted of macrophages with systemic hydrocortisone acetate (HA) and intratumor injections of antimacrophage serum (AMS) were examined 7 days after treatment. In control animals 30% of infiltrating host cells were esterase-positive tumor-associated macrophages (TAM) and 28.14% of tumor cells and 14.45% of endothelial cells were 3[H]TdR labelled. Hamsters treated with HA or HA and control serum showed no significant reduction in either the number of TAM or proportion of [3H]TdR labelled tumor of endothelial cells. AMS administered alone had no effect on either the content of TAM or 3[H]TdR labelling. In contrast hamsters treated with HA and AMS showed a 54% decrease in TAM and a 50% and 63% reduction in tumor and endothelial cell labelling respectively. These results suggest that growth and neovascularization of these tumors is mediated in part by macrophages.

Wound repair in mice as influenced by age and antimacrophage serum.

The closure of bilateral, full-thickness cutaneous wounds made over the back with a sharp paper punch was measured with calipers and assessed histologically in C57BL/6J male mice for 10 days after wounding. Young (6 months) mice exhibited a significantly more rapid rate of wound closure and repair than did mature (15 months) or aged (26 or 27 months) mice. The repair rate in mature and aged mice did not differ. Young mice, injected subcutaneously at the wound sites with rabbit antimouse macrophage serum (RAMMS) 5 min before wounding and on days 1 and 3 after wounding, exhibited slow delayed closure of cutaneous wounds during days 1 to 4 after wounding, similar to that of untreated aged mice. The early closure rate of mice injected with normal rabbit serum or physiological saline was rapid, resembling that of untreated young mice. The results suggest that cutaneous wound repair in mice is another physiological phenomenon whose rate of change is age related, but not necessarily progressive to senescence. The results also imply that macrophage functional decline may contribute to the slowing of wound repair in middle-aged and aged mice compared to young mice.

Biologic and Biochemical Characterization of a Macrophage-Derived Growth Factor for Rat Lung Fibroblasts

Growth factors such as platelet-derived growth factor (PDGF) and the transforming growth factors (TGF) have diverse biologic activities that influence physiologic development, wound healing and neoplastic transformation of cells. Macrophages have been shown to be important in the process of inflammation and repair through secretion of soluble mediators that can alter the growth of other cells. 1 Leibovich SJ Ross R The role of the macrophage in wound repair: a study with hydrocortisone and anti-macrophage serum. Am J Pathol. 1975; 78: 71-100 PubMed Google Scholar Studies in this laboratory with an animal inhalation model have shown that macrophages are attracted to sites of particle deposition by a complement-dependent mechanism and that these cells are temporally associated with a developing interstitial fibrotic lesion. 2 Warheit DB George G Hill LH Snyderman R Brody AR Inhaled asbestos activates a complement dependent chemoattractant for macrophages. Lab Invest. 1985; 52: 505-514 PubMed Google Scholar , 3 Warheit DB Hill LH George G Brody AR Time course of chemotactic factor generation and the corresponding macrophage response to asbestos inhalation. Am Rev Respir Dis. 1986; 134: 128-133 PubMed Google Scholar In the present study, we show that rat alveolar macrophages (AM) activated by particulates in vitro secrete a heat-stable competence factor for rat lung fibroblasts (RLF) with immunologic similarities to a sis oncogene-predicted synthetic peptide.

Blockade by antimacrophage serum of the migration of PMN neutrophils into the inflamed peritoneal cavity.

The effect of rat antimacrophage serum (rAMS) was tested on the influence of normal or thioglycollate-stimulated macrophage populations of the rat peritoneal cavity on the migration of polymorphonuclear neutrophils (PMN) induced by carrageenin, heterologous serum (rabbit) and sheep red blood cells. The rAMS used did not cross-react with PMN or lymphocytes nor did it affect circulating white cells, complement levels or lysed PMN present in the inflammatory exudate. It did, however, give a positive immunofluorescence reaction with resident and stimulated macrophages. The rAMS inhibited macrophage function as tested by sheep red blood cell phagocytosis in vivo and release of a PMN chemotactic factor(s) in vitro. Thioglycollate-stimulated peritoneal cavities showed an increased macrophage population and responded with increased PMN migration when challenged with heterologous serum or carrageenin, as compared with control rats. The presence of rat antimacrophage antibodies inhibited PMN migration induced by heterologous serum, sheep red blood cells and carrageenin. It is concluded that resident macrophages participate in the control of PMN migration to the site of an acute inflammation by acting as 'alarm cells' and triggering several defence mechanisms which ultimately protect the host from injurious stimuli.

The identification and characterization of antimacrophage serum (AMS) and its application to murine fibrous histiocytoma.

The preparation of antimacrophage serum (AMS) and its application to murine fibrous histiocytoma are reported. AMS, prepared by inoculating peritoneal macrophages from mice into rabbits, was purified by repeated absorption with kidney homogenate, red blood cells, thymus cells, non-adherent spleen cells and finally, L-cells. The molecular weight was determined as 170,000 daltons using SDS-polyacrylamide gel electrophoresis. AMS diluted to 1:32 reacted with 61.4% of peritoneal macrophages, but not red blood cells, lymphocytes, granulocytes and fibroblasts, as judged by membrane immunofluorescence. 57.1% of monoclonal B-10 macrophages and 75.6% of 28-12 macrophages transformed with SV40 reacted with AMS. B-10 and 28-12 cell tumors simulating human malignant fibrous histiocytoma gave a distinctive diffuse pattern of immunofluorescence with AMS which affected both round- and spindle-shaped cells, although positive fluorescence of spindle-shaped cells was somewhat weaker than that of round-shaped cells. However, tumor tissues from a murine fibrosarcoma showed no reaction with AMS. Based on these findings, AMS may be useful in determining the histogenesis of some soft tissue tumors of doubtful origin.

The effect of removal of adherent cells in lectin and allogeneic cell stimulation of bovine lymphocytes

Macrophages have been shown to play an important role in lymphocyte activation in many species but not unequivocally for bovine. In this study we have shown that when sufficient care is taken to remove macrophages by adherence or by anti-macrophage serum and complement, bovine lymphocytes respond poorly to lectins or to allogenic cells in mixed lymphocyte culture. Addition of macrophages to approximately the same level as found in the unseparated cells restores the proliferative responses. The degree of response shown by the macrophage depleted cultures depends on the lectin concentration. Stimulation with lower doses of lectin is more sensitive to macrophage concentration. The mixed lymphocyte response and the response to Concanavalin A (ConA) are more sensitive to macrophages than is the response to phytohemagglutinin (PHA).

Mechanisms of enhanced resistance of Mycobacterium bovis BCG-treated mice to ectromelia virus infection.

The mechanism of enhanced resistance of Mycobacterium bovis BCG-treated mice to ectromelia virus infection was investigated by determining the effect of splenectomy, antithymocyte serum, and antimacrophage serum on resistance. It was greatly reduced by these treatments, not only in normal mice, but also in mice treated with live or heat-inactivated BCG. Production of circulating interferon by ectromelia virus and Newcastle disease virus was augmented in BCG-treated mice and was markedly depressed by splenectomy and antithymocyte and antimacrophage serum treatments in both BCG-treated and normal mice. Carbon clearance activity was activated in BCG-treated mice, but splenectomy did not influence phagocytic activity. These results suggest that augmented interferon production in the spleens of BCG-treated mice plays a major role in enhanced resistance. Other possible mechanisms are discussed.

Increase of MLC sensitivity by elimination of a non-adherent responder cell subpopulation with anti-macrophage serum

A specific rabbit anti-rat macrophage antiserum (SAM) was prepared with a cytotoxic reactivity pattern complementary to that of a specific anti-lymphocyte serum. This was used to characterise adherent and non-adherent spleen cell subpopulations in mixed lymphocyte cultures. Adherent SAM + cells reacted as accessory cells whereas non-adherent SAM + cells were suppressors. Selective elimination thus achieved resulted in a highly significant increase of MLC reactivity in certain strain combinations and in conversion from non-reactivity to reactivity in others.

Surface antigens of murine hemopoietic stem cells. VIII. Antisera define lineage antigens held in common between granulocyte-macrophage cells and between lymphoid cells.

The cell-lineage model of hemopoietic cell differentiation has been further investigated by detailed absorption analysis of the anti-stem cell activity in rabbit antisera against mouse hemopoietic cells. Of seven differentiated hematopoietic cells tested, platelets alone absorbed the anti-stem cell activity in anti-platelet serum. Thymocytes and B-lymphocytes absorbed all of the anti-stem cell activity in antithymocyte serum whereas other nonlymphocytic cells showed only partial absorbing ability. Macrophages and granulocytic cells absorbed most of the anti-stem cell activity in antimacrophage serum and antineutrophil serum whereas other cell types showed little or no absorbing capacity. Antisera against a cloned mast cell precursor line showed partial cell lineage activity whereas anti-sera against eosinophils and B-lymphocytes showed no evidence of cell lineage activity. A detailed model of cell lineage antigens on hemopoietic cells is presented.

The role of mononuclear phagocytes in cardiac allograft rejection in the rat: I. Ultrastructural and cytochemical features

Mononuclear phagocytes (MNP) have been identified in rejecting rat cardiac allografts by morphological and cytochemical criteria. Their accumulation has been quantitated and their distribution within the graft recorded. Lymphocytes were the major infiltrating cell type present 3 days after transplantation, but by Day 5 and Day 7 there were 2.5 to 3 times as many MNP as lymphocytes. In the later stages (Days 6 and 7) many MNP were closely adjacent to myocardial cells and frequently possessed pseudopodia which were indenting the myocardial cell membrane. Allograft recipients given 750 rads γ-irradiation and reconstituted with thoracic duct lymphocytes rapidly rejected the graft. As many MNP were present in such grafts as in unmodified recipients. A potent antimacrophage serum did not prolong graft survival or alter the numbers of MNP within rejecting grafts. We conclude that MNP must be considered strong candidates for effector cells in allograft rejection and that satisfactory depletion techniques for MNP are not yet available.

Killing of the malarial parasite Plasmodium yoelii in vitro by cells of myeloid origin

Cytotoxic effects of mouse cells on Plasmodium yoelii were sought directly by incubating parasitized red cells with cells of various kinds for 16 h and then determining the percentage parasite survival in vivo, in terms of infectivity for the mouse. Cell populations rich in lymphocytes, e.g. lymph node and spleen, were less active than peritoneal cells and blood. Parasite killing by peritoneal cells was associated with macrophages: treatment with anti-macrophage serum (AMS) or depletion by adherence or centrifugation on Ficoll decreased activity. Polymorphonuclear leucocytes (PMN) in induced exudates may have contributed to killing, although not as actively cell for cell, and an effect of eosinophils in worm-induced exudates was not excluded. White blood cells were most active of all and fractionation on Ficoll confirmed that lymphocytes were relatively ineffective. The effector cell was phagocytic but it was insensitive to AMS. Tests on populations wih high or low proportions of PMN showed that parasite killing was independent of PMN number. It is concluded that the effector cell belongs to the monocyte-macrophage series and has acquired the ability to kill the parasite before becoming fully differentiated into a macrophage.

BCG-induced enhancement of endotoxin sensitivity in C3H/HeJ mice. II. T cell modulation of macrophage sensitivity to LPS in vitro

BCG infection induces a marked increase in LPS sensitivity in vivo and will render genetically defective, LPS hyporesponsive, C3H/HeJ mice almost as sensitive to LPS as normal mice. In this study, we have examined the endotoxin sensitivity of lymphocytes and macrophages from BCG infected mice in order to determine the cellular basis of this effect. We have found that the alteration in endotoxin sensitivity is mediated by a primary effect of BCG infection on T lymphocytes rather than on macrophages. Macrophages from «LPS sensitive», BCG-infected C3H/HeJ mice remain unresponsive to LPS when tested in vitro. However, when peritoneal T lymphocytes from these LPS »corrected» mice were cocultured with LPS unresponsive C3H/HeJ macrophages, a conversion to the LPS-responsive state was observed as manifested by the ability of the macrophages to produce LAF (IL 1) upon LPS stimulation. T cells from normally LPS-responsive or BCG-infected C3H/HeN mice, but not from control C3H/HeJ mice, were also able to render C3H/HeJ macrophages sensitive to LPS. This activity was not affected by treatment of the column-purified T cells with anti-macrophage serum plus complement, indicating that the response was not due to residual LPS-responsive macrophages contaminating the T cell preparations. The ability of the T cell suspension to render C3H/HeJ macrophages capable of producing LAF (IL 1) in response to LPS was abrogated by treatment of the T cell preparations with anti-Thy 1.2 plus complement. These findings establish the importance of T lymphocytes in regulating the LPS sensitivity of macrophages in BCG infected C3H/HeJ mice and support the concept that macrophage LPS responsiveness is dependent upon a certain state of macrophage activation which is regulated by lymphocytes.

Effects of prednisolone and anti-macrophage serum on the development of carditis and arthritis due to group A hemolytic streptococci in mice.

The effects of prednisolone and anti-macrophage serum (AMS) on the development of cardiac and articular lesions were compared in experimentally infected mice with group A hemolytic streptococci. The predonine-treated mice had a higher mortality rate and greater abscess formation than in the AMS-treated mice. The predonine-treated group had a higher incidence of cardiac lesions due to chronic pericarditis and chronic myocarditis, and the former was particularly frequent. Pathologic findings seen in the AMS-treated mice were those of acute pancarditis. The AMS-treated animals also had lesions of subacute or chronic arthritis, whereas articular lesions encountered in the Predonine-administered group were frequently those of chronic periarthritis and, in no instance, synovitis. These data indicated that treatment with AMS is more effective than that with prednisolone to experimentally induced rheumatic fever in laboratory animals.

Studies on the role of lymphocyte-activating factor (Interleukin 1) in antigen-induced lymph node lymphocyte proliferation

The in vitro proliferation of primed lymph node lymphocytes (LNL) in response to the soluble antigen ovalbumin (OVA) was dependent upon the presence of adherent cells. Restoration of OVA-induced LNL proliferation could be achieved by addition of highly purified lymphocyte-activating factor (LAF; Interleukin 1, IL 1): LAF (IL 1) did not stimulate LNL proliferation in the absence of the priming antigen or T lymphocytes. Furthermore, treatment of the LNL with antimacrophage serum completely blocked the ability of the LNL to respond to OVA and LAF (IL 1), suggesting that the residual macrophages in the LNL population were necessary to provide an additional function or signal, possibly antigen presentation, in conjunction with LAF (IL 1). These data therefore support the two signal hypothesis of macrophage-mediated lymphocyte activation and demonstrate the ability of LAF (IL 1) to provide one of these signals.

The role of the macrophage in Marek's disease: In vitro and in vivo studies

Macrophages form S- and K-strain Leghorn chickens, susceptible and restant to Marek's disease (MD) respectively, were studied to determine the macrophage contribution to the dynamics of MD infection, tumorigenesis and genetic resistance to this disease. In vitro studies demonstrated that macrophages from bothstrains were similar in their responses toward JM strain of Marek's disease virus (MDV) and JM-1 tumor cells. Macrophages were observed to phagocytize JM virus, but the interiorized virus was not seen to replicate within the macrophage or induce antigenic changes of the cell membrane. Clearance of JM-1 tumor cells was by both cytolytic and phagocytic mechanism. In vivo selective suppression of macrophage functions by antimacrophage serum or trypan blue inoculations resulted in significantly elevated viral titers and increased tumorigenesis, as compared to infected, non-suppressed or non-infected control groups. Results from this study indicate that genetic susceptibility or resistance to MD, as exhibited by S- and K-strain chickens, respectively, is not controlled at the macrophage level. The role of the macrophage in MD infection appears to be specifically surveillance.