Frank DeLustro

Mast cells and their degranulation in the Tsk mouse model of scleroderma

Abstract The Tsk mouse is a genetically transmitted example of cutaneous fibrosis which has been compared with human scleroderma. During a systematic histopathological study of the Tsk mouse, both an increased number and an increased proportion of degranulated mast cells were observed. The consistent association of mast cells and fibrosis in scleroderma, graft-vs-host reactions (GVHR), and now the Tsk mouse raises the question of a pathogenetic role for mast cells in fibrotic disorders in general.

Comparison of inflammatory changes in established type II collagen- and adjuvant-induced arthritis using outbred wistar rats

Type II collagen- and adjuvant-induced arthritis in outbred Wistar rats were compared using parameters that measured the inflammatory response, cellular and humoral immunity, blood protein changes, drug metabolism and histopathological and bony changes of the inflamed paws. There was a lesser incidence (40-70%) and severity of collagen disease than the adjuvant model (incidence approximately 100%). The use of MDP increased the incidence and severity of collagen arthritis. The acute phase protein response (plasma fibrinogen) was similar in both models during the peak of inflammatory response. Drug metabolism was inhibited in both type II collagen boosted with MDP or M. butyricum sensitized rats with arthritis; however, arthritic rats sensitized with collagen alone produced no inhibition. Only collagen arthritic rats produced type II collagen antibody and exhibited delayed hypersensitivity to type II collagen. Bony changes as assessed by radiographic evaluation were more severe in adjuvant arthritic rats than in the collagen arthritic model; histopathological findings from these animals confirmed this observation. The primary lesions in both models were periosteal reaction of the bone and ankylosis. Several classes of antiarthritic drugs were compared in both models using paw edema measurements and bony changes by radiographic evaluation. Drugs with inhibitory activity in both models were indomethacin, methylprednisolone, D-penicillamine and gold sodium thiomalate. Levamisole, chloroquine and auranofin were inactive in both models.

Human monocyte modulation of endothelial cells and fibroblast growth: Possible mechanism for fibrosis

Several human diseases are characterized by vascular pathology, fibroblast activation, and excessive fibrosis (e.g., scleroderma, chronic graft versus host disease, pulmonary fibrosis). An intense inflammatory exudate of mononuclear cells which are derived from the peripheral blood precedes the vascular and fibrotic changes. We examined, in vitro, the effects of human peripheral blood mononuclear cell culture supernatants (PBM-SN) on the growth and survival of human endothelial cells (EC) and of human dermal fibroblasts (FB). The same PBM-SN consistently induced inhibition of EC and stimulation of FB proliferation. PBM-SN derived from 42 patients with scleroderma induced 32 +/- 5% (mean +/- SE) more inhibition of EC and 42 +/- 18% more stimulation of FB compared with PBM-SN derived from 30 healthy subjects. Depletion of phagocytic cells or adherent cells from PBM resulted in SN with no demonstrable activity on either EC or FB. Partial purification of PBM-SN on ion exchange and gel filtration chromatography revealed the presence of two fractions that stimulated and one fraction that inhibited FB proliferation, and two fractions that inhibited and one that stimulated EC proliferation. These data suggest that monocytes are capable of releasing mediators that stimulate or suppress EC or FB. However, when activated by surface adherence, resulting SN inhibit EC and stimulate FB proliferation. Serum is required for the expression of FB proliferation.

Adoptive Transfer of tsk Skin Fibrosis to +/+ Recipients by tsk Bone Marrow and Spleen Cells

Abstract The tight skin mouse (tsk/+) is an autosomal dominant example of inherited fibrosis whose pathogenesis is unclear. Autoimmune phenomena have been described previously. This study demonstrates the adoptive transfer of skin fibrosis to lethally irradiated syngeneic +/+ recipients by the transplantation of both bone marrow and spleen cells. The recipient skin fibrosis was not associated with mast cell proliferation or degranulation as it is in the tsk/+ mouse, representing for the first time a dissociation between mast cells and fibrosis and introducing the potential for direct T cell-fibroblast interactions.

Immune Response to Connective Tissue Components of the Basement Membrane

The immune response to connective tissue components of basement membrane (type IV collagen and laminin) and to interstitial collagen (type I) has been examined in human and murine systems. We also examined the role that immunologic sensitization to autologous connective tissue components might play in inducing an inflammatory response resulting in pathologic sequelae. Mice receiving a single subcutaneous injection of 5 micrograms type IV or type I murine collagens, or murine laminin in complete Freunds adjuvant mount a delayed-type hypersensitivity response characterized by a mononuclear cell infiltrate when challenged in the footpad with the sensitizing antigen. Cell-mediated immunity to these connective tissue antigens can be transferred to normal syngeneic mice with sensitized T-lymphocytes. In addition, repeated immunizations with these homologous connective tissue components elicit antibody responses in mice. Our data demonstrate the immunogenic nature of types IV and I collagen, and of laminin in a syngeneic murine model. We have demonstrated autoantibodies to the basement membrane and interstitial collagens in the sera of patients with scleroderma (systemic sclerosis); ELISA ratios correlate directly with the extent of pulmonary fibrosis in these patients. Anti-type IV collagen autoantibodies were found to be primarily IgM and anti-type I collagen antibodies, primarily IgG. An antibody response to autologous connective tissue antigens could lead to complement activation, immune complex formation, and deposition of the complexes along vascular endothelium with recruitment of blood monocytes in situ, mirroring the early scleroderma lesion (perivascular mononuclear cell filtrates). In vitro we examined the role of human peripheral blood mononuclear cells in the activation of fibroblasts. Adherent human blood monocytes release mediators which stimulate fibroblast proliferation and collagen deposition. A model is presented for the induction of immunity to autologous connective tissue components, leading to mononuclear cell inflammation, fibroblast activation and fibrosis. Selective immunity to basement membrane collagens may influence the clinical expression of diffuse connective tissue syndromes such as scleroderma (systemic sclerosis).

Effect of gold salts, D-penicillamine and benoxaprofen on type II collagen-induced arthritis in rats

We investigated the therapeutic effect of two gold salts, gold sodium thiomalate (GST, i.m.) and auranofin (p.o.),d-penicillamine (p.o._ and benoxaprofen (p.o.) in rat collagen-induced arthritis using type II collagen from fetal bovine articular cartilage.GST, but not auranofin, reduced hind paw edema and bone pathology. However, auranofin reduced serum copper and zymosan-induced prostaglandin production from peritoneal macrophages. In contrast, GST increased both serum copper and macrophage prostaglandin production by zymosan.Benoxaprofen reduced both hind paw edema and pathology, whereasd-penicillamine was without effect. None of these treatments influenced the circulating level of antibody to type II collagen.

Cell-mediated immunity to homologous basement membrane (type IV) collagen in C57BL6 mice

Abstract We have studied induction of a cell-mediated immune response to homologous type IV collagen in C 57 BL 6 mice, following a single sensitizing exposure. Antigens were characterized by amino acid composition and SDS-gel electrophoresis, comparing immunogens with previously characterized standard collagens. The delayed-type hypersensitivity (DTH) response was assayed after sensitizing mice with pepsin-treated type IV collagen (TIVp) in Freunds complete adjuvant (FCA), Freunds incomplete adjuvant (FIA), or PBS by measuring footpad swelling at various time intervals following challenge with soluble collagen preparations. Mice sensitized with TIVp in FCA or FIA developed a peak DTH response on Day 7 at 24 hr after challenge with TIVp. Mice immunized with TIVp alone demonstrated a delayed peak DTH response 11 days after sensitization and displayed a significantly lower degree of reactivity. No evidence of an immediate hypersensitivity response was detectable at 4 hr in any group sensitized with TIVp. No group challenged with type 1 collagen or pepsin-treated type I collagen exhibited significant footpad swelling throughout these experiments; normal and adjuvant control mice failed to show significant DTH. Histology of the footpad lesions of TIVp-sensitized mice on Day 7. 24 hr after challenge with TIVp, revealed an infiltration of mononuclear cells characteristic of a DTH response. Passive transfer of 25 × 106 sensitized spleen cells at Day 7 post-TIVp injection into normal mice resulted in significant DTH responsiveness to challenge with TIVp after 48 hr; T-cell-depleted cells from TIVp-sensitized spleen failed to transfer significant DTH. These studies demonstrate the immunogenic nature of type IV collagen in a syngeneic murine model.

Delayed-type hypersensitivity to elastase-soluble lung peptides in the tight-skin (Tsk) mouse☆

The development of immunity to homologous connective tissue antigens was studied with respect to aging in the tight-skin (Tsk) mouse mutant. A delayed-type hypersensitivity (DTH) response to elastase-solubilized lung peptides in Tsk/+ mice, which became evident at 10 weeks of age and increased in intensity until 22 weeks, was observed. Tsk mice did not demonstrate significant DTH responses when challenged with type I or IV collagen, and normal (+/+) littermates of all ages did not respond to any of the antigens under study. DTH responses could be adoptively transferred to normal +/+ and C57BL/6 mice with spleen cells from 30-week-old Tsk/+ mice; treatment with anti-Thy 1.2 antibodies plus complement significantly reduced the ability of these Tsk/+ cells to transfer DTH reactivity. No antibody activity to the antigens under study could be detected in the sera of Tsk/+ or +/+ mice at any age. These results are discussed with regard to the pathological manifestations observed in the Tsk/+ mutant mouse.

Immune response to laminin, a noncollagenous glycoprotein of basement membrane, in a syngeneic murine system.

Abstract The immune response to the noncollagenous, attachment glycoprotein of basement membrane (laminin) was studied in a syngeneic murine system. Delayed-type hypersensitivity (DTH) was assayed in C57BL/6 mice by measuring footpad swelling following challenge with connective tissue antigens. Mice receiving a single sensitizing injection of laminin in Freunds complete adjuvant (FCA) developed a significant DTH response which peaked on Day 7, 24 hr after challenge with laminin or collagenase-treated laminin. Laminin-sensitized mice failed to show any significant footpad swelling when challenged with types I or IV collagen, or fibronectin throughout these experiments. Normal mice displayed no significant DTH when challenged with these collagenous or noncollagenous connective tissue antigens. Adoptive transfer of laminin-sensitized spleen cells into normal mice resulted in significant DTH responsiveness to challenge with laminin; depletion of T cells from the immune spleens abrogated this response. Twenty-four hours after challenge with laminin, histology of the footpad lesions of laminin-sensitized mice revealed a mononuclear cell infiltrate, characteristic of a DTH response. Mice receiving repeated injections of laminin in Freunds incomplete adjuvant (FIA) developed significant antibody responses as detected by the enzyme-linked immunosorbent assay (ELISA). Furthermore Pronase, but not collagenase, treatment of laminin destroyed its antigenicity. Laminin immune sera showed no reactivity when assayed on fibronectin or collagen types I-V. No cross-reactivity was exhibited by murine anti-type IV or anti-type I collagen antisera with laminin. These studies demonstrate the ability of isologous laminin to induce antigen-specific cell-mediated and humoral immunity in a murine model.

The absence of antibodies to type II collagen in established adjuvant arthritis in rats

Utilizing an adjuvant arthritis model in rats, we examined humoral immunity to collagen and inflammation in animals with active disease and during drug therapy. Humoral immunity to types I or II collagen was not detected in the sera of rats with advanced adjuvant arthritis; this was in marked contrast to rats with type II collagen-induced arthritis which possessed serum antibodies to native and denatured type II collagen. Hind paw edema and bone pathology were monitored as parameters of inflammation. A new investigational drug, Wy-41,770, was most effective in reducing all of these aspects of inflammatory disease while indomethacin, methylprednisolone, andd-penicillamine caused a less significant diminution of only some of these parameters of inflammation. Antibodies to collagen were not detected in the sera of rats treated with the drugs under study. These data demonstrate that adjuvant arthritis can occur in rats in the absence of antibodies to types I or II collagen.