G. Waldmann

Significance of cell-mediated and humoral immunity in the acute and chronic phase of antigen-induced arthritis in rabbits

In the course of antigen-induced arthritis of rabbit cell-mediated and humoral immune responses were repeatedly tested in order to prove their significance for the acute and chronic phase of inflammation. The arthritis was monitored during the progression of the inflammation by means of the joint swelling and at the end of experiments by histological evaluation of synovitis and cartilage degradation. Following the arthritis induction a strong increase of specific antibodies and of circulating immune complexes was evident. The correlations between antibody levels and joint swellings confirmed that the local formation of immune complexes is responsible for the initiation and perpetuation of arthritis. In the early phase after immunization the responsiveness of lymphocytes to antigenic and mitogenic stimulation was increased, in the late chronic phase of arthritis proliferative responses of lymphocytes to cartilage matrix components were revealed. No direct correlations could be demonstrated between any cell-mediated immune response and the severity of arthritis. The hyperreactivity of cell-mediated immunity is suggested to be responsible for the transition of the acute arthritis into the chronic stage. The deficiency of an effective suppression results in the activation of B-lymphocytes with increased production of antibodies, maintaining the inflammatory process for a long time. Under these conditions the release of cartilage matrix components during the acute joint reaction induces autoimmune responses against cartilage, which could contribute to the chronification of arthritis and to cartilage degradation.

CHANGES OF IMMUNOREGULATORY PROPERTIES AND INDUCTION OF AUTOIMMUNE REACTIVITY TO CARTILAGE IN RABBITS WITH ANTIGEN-INDUCED ARTHRITIS

The chronicity of the antigen-induced arthritis is characterized as dependent on the development of cell-mediated immunity to the antigen, but the exact mechanisms underlying are unclear. We have evidenced decreased suppressor and increased helper cell potential in the early phase of arthritis as result of the immunization procedure. In the late phase of arthritis proliferative responses of spleen lymphocytes to cartilage proteoglycans were revealed which were neither present in immunized animals without arthritis induction nor in the early phase of arthritis. The changes of the regulatory properties on the T-cell level are probably responsible for the transition of acute arthritis into the chronic stage. The deficiency of an effective suppression and/or the increased helper cell potential results in the activation of B- and T-lymphocytes with increased cell-mediated and humoral immune responsiveness to the antigen maintaining the inflammatory process for a long time. In this situation the release of cartilage proteoglycans during the acute joint reaction induces autoimmune responses against cartilage which could contribute to the chronification of inflammation and to cartilage degradation.

Effects of the immunomodulator diacetyl-splenopentin on antigen-induced arthritis in rabbits

Long-term treatment with the immunomodulator diacetyl-splenopentin reduces the severity of chronic joint inflammation and cartilage destruction in rabbits with antigen-induced arthritis. The level of specific antibodies as well as specific and non-specific cell-mediated immune reactivities including the proliferative response of spleen lymphocytes to cartilage proteoglycans in treated animals are lower than in untreated arthritic rabbits. Moreover, suppressor cell activity, which normally decreases during the early phase of inflammation, is enhanced and hyperreactive helper cell potential is reduced. These findings suggest that treatment with diacetyl-splenopentin normalizes the immune regulation, which is disturbed in the early phase of inflammation. This might result in a depression of the hyperreactive immune system including the autoimmunity developed against cartilage. Lowered immune reactivity in the joint in turn reduces the severity of chronic joint inflammation.

Studies on a new acute phase protein: I. Immunocytochemical demonstration of the origin

Synovial fluid of patients with rheumatoid arthritis or traumatic arthritis contains an antigen which is thermostable to boiling temperature and insoluble in ethanol. The antigen was not found in sera of healthy subjects but it is present in numerous sera of patients with different inflammatory diseases. The partial purification of the antigen and the production of specific antisera are described. Immunofluorescent staining of tissue sections and blood smears indicates, that the antigen is a cytoplasmic protein of polymorphonuclear leucocytes. Monocytes contain the antigen to a lower degree, it was not found in eosinophils and lymphocytes. In addition, the influence of different fixing agents and of other pretreatment on the pattern of the cell fluorescence was studied. We propose to designate the antigen as thermostable granulocyte antigen (TSGA).

Lectin induced experimental arthritis in rabbits.

Summary Lectins as substances of plant origin are capable to react specifically with certain caroDhydrate components of cell and tissue constituents, especially of connective tissue structures. By a single injection of Lens culinaris lectin (LcL) into the knee joint of rabbits an experimental arthritis could be produced. In comparison to earlier communications, in this paper the duration of the LcL-induced arthritis in the chronic phase as well as the effect of some other lectins as Pisum sativum lectin (PsL), Concanavallin A (Con A) and Wheat germ agglutinin (WGA) was studied with special reference to the pathogenetic mechanisms. After intraarticular application of the lectins a prolonged dose dependent arthritis developed with the tendency of chronification. The histomorphological feature of the persistant arthritic process was characterized by lymphoplasmocytic infiltration, fibroblast and histiocyte proliferation, proliferative vasculitis and villous hyperplasia of the synovial membrane, at all. The arthritis lasted up to 6 months after a single injection of LcL and was strongly raised by a following second injection. Different lectins resulted in quantitatively different changes the slightest of which were induced by the non-mitogenic WGA. As to the pathogenesis, we consider the high binding affinity of the lectins to the structures of connective tissues with long-time persistance of the substances at the site of injection to be of fundamental importance, further on the mitogenic activity of lectins may lead to unspecific stimulation of T-lymphocytes with release of biologically active substances like mediators of cellular immunity and induction of cytotoxic effects, and at last the lectins as plant proteins initiate a humoral immune response with tissue bound antigen antibody reaction and possible specific as well as non-specific complement activation.

Autoreactive T-helper cells in experimental arthritis

Experimentally induced arthritis models have been widely used in order to study and test antiinflammatory and antiarthritic drugs and, furthermore, to investigate pathogenic mechanisms of joint inflammation. Most of the chronic arthritides in animals are initiated immunologically, i.e. antigeninduced arthritis (AIA), adjuvant arthritis (AA) and collagen type H-induced arthritis (CIA) [1-3]. Moreover, MRL-lpr mice which are characterized by severe immunopathy develop spontaneously a rheumatoid arthritis (RA)-like polyarthritis [4]. The AIA is induced by injection of an antigen into the knee joint of animals preimmunized with the same antigen in complete Freunds adjuvant (CFA). A short acute reaction, mediated by formation of immune complexes, is followed by a chronic inflammation, which persists for a long time and shows histopathologically similar features to human RA. For the development of the chronic phase cell-mediated immunity is essential, but the exact mechanisms responsible are not completely clear. Recently, we have found a strong increase in antibody levels after the intraarticular injection of the antigen and an increased responsiveness of cultured lymphocytes to antigenic and mitogenic stimulation in the early phase of AIA in rabbits [5]. Now we show the activities of immunoregulatory T-cells and autoimmune reactions against connective tissue constituents during the progression of arthritis. Materials and methods

Biphasic changes of the immunological reactivity in the course of experimental lectin-induced arthritis of rabbits

A single injection of Lens culinaris lectin (LcL) into the knee joint cavity of non-sensitized rabbits produces an arthritis with an acute and chronic phase, lasting up to one year. The persistence of the lectin in the joint, related to the strong binding affinity of lectins to glycoproteins of connective tissue structures, and the presence of specific antibodies against LcL in the serum after the intra-articular injection make this model comparable to the antigen-induced arthritis. But in our system these conditions are further modified or amplified by the mitogenic activity of LcL itself. The cell-mediated immunity, studied by mitogenic stimulation of peripheral blood lymphocytes, is characterized by a biphasic change in the course of this experimental arthritis. Hyperresponsiveness to stimulation with LcL and Concanavalin A (Con A), decreased Con A-induced suppressor cell activity, and stimulatory serum factors could be detected in the early phase of inflammation. The late phase of arthritis (8 months after the induction) was characterized by hyporesponsiveness to mitogenic stimulation, normal suppressor cell activity and inhibitory serum factors. In spite of the differences of this experimental arthritis to the human rheumatoid arthritis, concerning mainly the initiation and the lack of systemic manifestation, there are surprising similarities between both, not only in the histopathological feature and the chronicity but also in the cell-mediated immune reactions. Therefore, similar pathogenetic mechanisms for the chronic phase can be suggested.

Killing of untreated target cells by nonimmunized rat macrophages in vitro: II. Ultrastructural studies on the interaction of macrophages and rat liver cells

The sequence of morphological changes following the interactions between macrophages of nonimmunized rats and allogeneic liver cells in vitro were examined by scanning and transmission electron microscopy. The effector cells could be identified as macrophages because of their morphologic characteristics and their ability of latex beads incorporation. The cytotoxic reactions required close contracts between the reactive cells. Effector membrane processes and microvilli attached the target cell surface. Broad contacts and, more frequent, point contacts were observed. After 4 to 5 hrs of incubation the target cells showed pinocytotic activities in the contact regions. Later on submembraneous cytoplasmatic leaks or defects indicated the increasing damage of the target cells. Finally, liver cell ghosts with degenerated organelles were found as morphologic expression of general target cell destruction whereas effector macrophages did not show any damage during the cytotoxic reaction.

Killing of untreated target cells by nonimmunized rat macrophages in vitro. I. Studies on the type of effector cells and their capacity to destroy allogeneic target cells.

Spleen cells derived from unsensitized Wistar rats lysed untreated, allogeneic liver cells of high passages (93rd until 159th subculture) as well as allogeneic tumor cells in mixed cultures. On the other hand, rat liver cells between passages 42 and 56 and allogeneic or xenogeneic fibroblasts were not destroyed by rat spleen cells in vitro. The degree of target cell lysis was dependent on the incubation time and effector-target cell ratio. In the rat spleen, the macrophages were detected as effector cells. Rat peritoneal cells showed the same effects as rat spleen cells. Trypsin treatment caused no loss of cytotoxicity of rat macrophages. Thymocytes and blood lymphocytes of rats were not cytotoxic against allogeneic or xenogeneic target cells. The macrophages apparently destroyed target cells by a nonphagocytic form of cell contact. Unstimulated spleen cells of guinea pigs, Syrian hamsters, CBA mice and rabbits were not capable to destroy target cells in allogeneic and xenogeneic systems.

Demonstration of lipopolysaccharide (LPS) binding sites on the cell surface of guinea pig peritoneal macrophages by means of immunogold technique

Lipopolysaccharide binding sites of guinea pig peritoneal macrophages were demonstrated by means of immunogold technique. Resident peritoneal macrophages show a strong specific binding of bacterial lipopolysaccharides from E. coli to cell surface structures.