Anna Ridderstad

Xenograft rejection of porcine islet-like cell clusters in normal and natural killer cell-depleted mice

Fetal porcine islet-like cell clusters (ICC) were transplanted under the renal capsule of normoglycemic normal or athymic (nu/nu) C57BL/6 mice. Control animals were implanted with allogeneic minced kidney tissue from C57BL/Ks mice. The animals were killed 6 or 14 days after transplantation and the grafts were processed for flow cytometric analyses or immunohistochemistry. Xenograft destruction was evident in normal mice on day 6 after transplantation. The majority of infiltrating cells were macrophage-like cells expressing the F4/80 antigen. Lymphocytes expressing the CD3 antigen were in minority and mainly located in the peripheral parts of the ICC xenograft. The frequency and distribution of CD4+ cells were found to resemble those of the CD3+ cells. A large number of infiltrating cells, including several macrophage-like cells, expressed the Thy 1.2 antigen. Flow cytometry of infiltrating cells in the ICC xenograft revealed that approximately half of the cells expressing the F4/80 antigen also expressed Thy 1.2 and/or CD4. No cells were found expressing both the F4/80 and CD8 antigens. Both the F4/80 single-positive and the F4/80, CD4 double-positive cells were found to be larger and more granular than the CD4 single-positive cells. No co-expression of CD4 or Thy 1.2 with the F4/80 antigen was detected on cells infiltrating allogeneic tissue grafts. Moreover, a relative large number of cells (approximately 15%) in the xenograft expressed the NK 1.1 antigen as determined by flow cytometry. The role of natural killer (NK) cells in islet xenograft rejection was further evaluated in mice depleted of NK cells, using intraperitoneal injections of the monoclonal antibody NK 1.1. The simultaneous inoculation and subsequent growth of the NK cell-sensitive beta 2-microglobulin-deficient mutant, C4.4-25-, lymphoma cell line EL-4 served as an in vivo control of NK cell depletion. However, all NK cell-depleted mice rejected the ICC xenograft. In contrast, athymic mice permanently accepted the porcine ICC xenograft but, readily rejected the NK cell-sensitive lymphoma cell line. Taken together, ICC xenograft rejection in mice seems to be T cell dependent, as evidenced in the nude mice model, while the main effector cell appears to be a macrophage with a unique phenotype.

Xenograft rejection of porcine islet-like cell clusters in immunoglobulin or Fc-receptor g-deficient mice.

The aim of the present study was to evaluate the role of xenoreactive antibodies in islet-like cell cluster (ICC) xenograft rejection. For this purpose, normal mice, mice with a targeted disruption of the Fc-receptor (FcR) gamma-chain, or the membrane exon of the immunoglobulin mu-chain gene, were transplanted with fetal porcine ICC under the kidney capsule. Mice lacking the FcR gamma have no functional FcR for IgG or IgE. Mice with disruption of the immunoglobulin mu-chain cannot produce antibodies, because B cell development is arrested at the stage of preB cells. All animals, irrespective of recipient group, readily rejected the ICC xenograft. Analyses of the pattern of cellular infiltration revealed only minor dissimilarities between the different experimental groups. Xenograft destruction was evident on day 6 after transplantation, and a large number of mononuclear cells were found to be evenly distributed throughout the ICC graft. The majority of the infiltrating cells were large, macrophage-like cells expressing the macrophage-specific phenotype marker F4/80. CD3-positive T lymphocytes were found to be mainly accumulated in the peripheral parts of the ICC xenograft. This study has demonstrated that xenoreactive antibodies are not crucial to ICC xenograft rejection in the pig-to-mouse model.

Immunological Characteristics of Islet Cell Xenotransplantation in Humans and Rodents

If ethical, physiological and immunological problems can be solved, it is conceivable that the pig will be a future donor species for human xenotransplantation (Cooper et al. 1991). The pig is a discordant species which implies that all humans have natural antibodies reactive against determinants present on most porcine cells. These determinants are present mainly on glycolipids and glycoproteins present on both nucleated cells and on erythrocytes. Most probably, these determinants are oligosaccharide residues, with structures similar to ABO-blood group substances (Szymanowski et al. 1926, Good et al. 1992, Holgersson et al. 1992, Koren et al. 1993). Porcine cells are sensitive to human complement, hence, porcine tissues can be immediately destroyed by antibody-mediated reactions, such as direct cytotoxicity. Natural antibodies react with determinants present on blood leukocytes, endothelial cells and other cells in a xenogeneic graft. Therefore, discordant vascularized grafts are hyperacutely rejected. However, not all cells are sensitive to the cytotoxic effect of antibodies. Sensitivity is in part determined

Synovial fluid from rheumatoid arthritis patients induces polyclonal antibody formation in vivo.

Our previous studies demonstrated the presence of a T‐cell replacing factor in the synovial fluid (SF) of patients with rheumatoid arthritis (RA) and that RA‐SF can activate, selectively, the induction of IgG2b antibody secreting cells, in lipopolysaccharide (LPS)‐pretreated mouse spleen cell cultures. In the present study the effect of RA‐SF was tested in vivo in mice. Injection of the polyclonal activator LPS induced the production of IgM and IgG3 secreting cells in normal mice. However, the addition of RA‐SF led to a selective increase in the production of IgG2b with a peak response on day Sand IgG1 plaque‐forming cells (PFC) with a peak on day 7. Neither the IgG2b nor IgG1 responses were caused by specific immunity against heterologous proteins present in RA‐SF, as injection of in vitro inactive RA‐SF samples did not induce PFC. The effect on B cells of RA‐SF was further evaluated by injection of RA‐SF in combination with LPS to the Xid B‐cell deficient CBA/N mice, RA‐SF had identical effects in CBA/N as in normal mice. The biological implication of these findings is discussed. Our earlier results support the idea that B cells are endogeneously activated in RA patients, We have speculated that this activation is caused by the B‐cell differentiation factor which is present in SF. Therefore, we also tested whether RA‐SF could influence antibody‐forming cells in mice that spontaneously develop autoimmunity. We found that injection of RA‐SF alone, in the absence of any other activating substance, induced a very marked increase of IgG producing cells in (NZW × NZB) F1 hybrid mice. From a relatively high background level the RA‐SF could still induce an up to 100‐fold increase in the numbers of PFC in spleens of such mice.

Rheumatoid Synovial Fluid Reconstitutes the B-Cell Defect in CBA/N Mice

Synovial fluid from patients with rheumatoid arthritis (RA‐SF) contains a biological activity which can replace T cells for activation of antibody secretion in human blood lymphoid cells and which can also induce the selective differentiation of IgG2b‐secreting cells in lipopolysaccharide (LPS)‐pre‐activated mouse spleen cells. The B‐cell activity of this factor was studied in CBA/N mice which have an X‐linked B‐cell immunodeficiency which manifests itself as a defective humoral response to certain thymus‐independent antigens (TI‐2). RA‐SF has now been shown to reconstitute partly the B‐cell deficiency in CBA/N splenic B cells in vitro Addition of RA‐SF to LPS‐pretreated cell cultures results in IgG2b secretion in CBA/N spleen cells as well. In contrast to cells from normal CBA mice, cells from CBA/N mice cannot respond to interleukin 4 (IL‐4) after addition of LPS with production of IgG1 antibodies in vitro. However, the addition of RA‐SF completely restores a normal IL‐4‐induced IgG1 response. No other biologically active factors have been shown to allow the production of IgG antibody producing cells in CBA/N splenic B cells. It is postulated that the xid immunodeficiency could be the result of a deficient production of a biological activity which is abundant in RA‐SF.

IgG2b Inducing Factor from Rheumatoid Arthritis Synovial Fluid Synergizes with Transforming Growth Factor-beta in Promoting IgG2b Antibody Production in Mouse B Lymphocytes

Rheumatoid arthritis synovial fluid (RA‐SF) contains a distinct biological activity that selectively induces IgG2b production in LPS‐activated murine B blasts. This IgG2b inducing factor (IgG2bIF) acts directly on purified LPS‐activated B blasts from normal and Brutons tyrosine kinase‐defective CBA/N mice. In order to test the possibility that TGF‐β and IgG2bIF in RA‐SF act in concert to induce IgG2b production, anti‐TGF‐β monoclonal antibodies and RA‐SF were added to LPS‐activated CBA B blasts, which led to a marked reduction of IgG2b‐producing cells. This result indicates that TGF‐β and IgG2bIF in RA‐SF synergize in the induction of IgG2b production. TGF‐β antibodies do not inhibit IgG2b production in CBA/N B blasts, further substantiating our earlier notion that CBA/N B blasts have a higher endogenous production of TGF‐β after LPS stimulation, which might be responsible for the aberrant reactivity in btk deficient CBA/N mice. RA‐SF is able to reconstitute the deficient IgG1 response in LPS‐ and IL‐4‐stimulated CBA/N B blasts. Addition of antibodies against TGF‐β had only a marginal effect on the IgG1 response, indicating that the reconstitution is mediated by another factor(s), which is present in RA‐SF.

Partial Biochemical Characterization and Purification of IgG2b Inducing Factor as a New Cytokine from Synovial Fluid of Patients with Rheumatoid Arthritis

Rheumatoid arthritis synovial fluid (RA‐SF) contains a novel biological activity, which selectively induces IgG2b antibody production in lipopolysaccharide (LPS)‐activated mouse spleen cells in vitro and in vivo. Our previous studies have shown that this activity is not functionally identical to otherwell‐known cytokines and interleukins. In this study we demonstrate the partial purification and biochemical characterization of the IgG2b inducing activity in RA‐SF. Biochemical characterization revealed that the IgG2b inducing activity in RA‐SF has the following properties: it is a protein, sensitive to pH > 11 and < 4, which is precipitated by 50% of saturated ammonium sulphate and has a molecular weight of 50–70 kDa; it binds to Cibacron‐blue and heparin and its activity is not mediated by immunoglobulins or immune complexes, which are present in RA‐SF. Biochemical characteristics of the IgG2b inducing activity also differ from other cytokines and interleukins. The term IgG2b inducing factor is proposed for this novel activity.

Rheumatoid arthritis synovial fluid enhances T cell effector functions

Rheumatoid arthritis is a chronic autoimmune joint disease of unknown etiology. T cells are believed to be important in the pathogenesis of rheumatoid arthritis since they infiltrate the joints and express several activation markers, such as MHC class II and IL-2R. In this study we have elucidated the effect on freshly isolated T cells of rheumatoid arthritis synovial fluid (RA-SF), which contains in vivo produced cytokines and enzymes. The mouse mixed lymphocyte culture (MLC) has been used as a model and specific cytotoxicity was evaluated against 51Cr-labelled sensitive target cells. Studies have shown that RA-SF contains a B cell differentiation activity that can cross-react between the human and murine species. Here we have shown that the addition of RA-SF strongly potentiates cytotoxic activity as well as lymphokine production by allogeneic activated effector T cells. The enhanced cytotoxicity induced by RA-SF was found to be due to a combined effect of increased cytotoxic T lymphocyte (CTL) precursor frequency, measured by limiting dilution analysis, and a more efficient killing on a per cell basis. Kinetic studies show that RA-SF must be added within 48 h after initiation of the MLC, otherwise the effect is lost. The target cell specificity of RA-SF was studied, using enriched CD4+ or CD8+ responder cells in the MLC. It was found that RA-SF could act directly on the CD8+ cells and potentiate their development to cytotoxic effector cells: this activity was not found when CD4+ responder cells were used instead. RA-SF could, on the other hand, greatly enhance IL-2 production by CD4+ responder cells. We suggest that B and T cell activity in RA-SF is important in the propagation of chronic inflammation in the joints of patients with rheumatoid arthritis.