Increased ICAM-1 expression in the early stages of murine chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is considered to be a model for scleroderma, and vascular changes are considered to be important in that disease. We have examined the expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function associated antigen-1 (LFA-1) using monoclonal antibodies and immunohistochemistry in very early murine cGVHD. ICAM-1 is found on a number of cell types including endothelial cells. It is the natural ligand for LFA-1 found on leukocytes. Adherence of leukocytes to ICAM-1 positive cells is mediated by LFA-1 and this binding is thought to play an important role in a number of cell adhesion events in immune reactions. Experimental cGVHD across minor histocompatibility barriers is established by the iv inoculum of B10.D2 spleen cells into a sublethally irradiated BALB/c host. Ear and skin biopsies were taken at Days 0-5 and from Days 14 to 120 postinoculum. In comparison to the control group (BALB/c spleen cells given to a sublethally irradiated BALB/c host), the cGVHD mice show increased ICAM-1 expression by Day 3 on endothelial cells and mononuclear cells (MNC) and on fibroblast-like cells by Day 4. By Day 14, there are increasing numbers of ICAM-1-expressing cells and increased epidermal reactivity for ICAM-1; and finally, an increased number of LFA-1 positive infiltrating MNC. These changes wane by Day 28 and are gone by Day 120. These results support the concept that ICAM-1/LFA-1 interactions play a role in the immune regulation of cGVHD. The very early upregulation of ICAM-1 on the endothelium indicates that this cell type may be playing a primary role in cGVHD, and this model should provide a simple system in which to test regulation of endothelial activation in vivo.

Antinuclear antibodies and breast implants.

Anecdotal reports have linked silicone breast implants to autoimmune diseases, with scleroderma being mentioned most often. In view of other environmental influences--such as silica mining and the toxic oil syndrome--thought to predispose to scleroderma, more systematic immunologic information on women with silicone implants is needed to assess any possible relation to autoimmunity. A cross-sectional survey was carried out with 150 women, of whom 131 had implants. Group 0 consisted of 19 volunteer women without breast implants who felt healthy, group I was 38 volunteer women with breast implants who felt healthy, group II was 82 women with implants who had various symptoms, and group III was 11 women with implants who had autoimmune disease. Scleroderma was overrepresented in group III (6 of 11). Antinuclear antibodies were determined on Hep-2 cells, using serum dilutions of 1:16, 1:64, and 1:256. A positive test was indicated by 1+ or more fluorescence at 1:256. Antinuclear antibody tests were positive in 0% of group 0, 18% of group I (P < .05 versus group 0), 26% of group II, and 64% of group III. There was no correlation between antinuclear antibody positivity and type of implant, indication for implantation, time since first implantation, total number of implants, and report of implant leak or rupture. Women with breast implants may be at risk for the development of antinuclear antibodies. Proof of such an association will require large-scale prospective studies and epidemiologic analyses.

Transplantation of enriched CD34-positive autologous marrow into breast cancer patients following high-dose chemotherapy: influence of CD34-positive peripheral-blood progenitors and growth factors on engraftment.

PURPOSE: To evaluate the capacity of enriched CD34-positive (CD34+) progenitor cells to reconstitute hematopoiesis in poor-prognosis breast cancer patients following administration of a high-dose alkylating agent chemotherapy regimen. PATIENTS AND METHODS: Forty-four breast cancer patients received high-dose chemotherapy followed by autologous bone marrow support (ABMS) with CD34+ hematopoietic progenitor cells in five sequentially treated cohorts. Following infusion of CD34+ marrow, cohort no. 1 received no growth factor, cohort no. 2 received granulocyte colony-stimulating factor (G-CSF), and cohort no. 3 received granulocyte-macrophage colony-stimulating factor (GM-CSF). Cohort no. 4 received the CD34+ fractions of both marrow and peripheral-blood progenitor cells (PBPCs) plus G-CSF. Cohort no. 5 received only the CD34+ PBPCs plus G-CSF. Immunohistochemical staining for breast cancer was performed on all hematopoietic cell products before and after the positive selection procedure, to assess quantitatively the level of tumor-cell contamination. RESULTS: Cohorts no. 1, 2, 3, 4, and 5 achieved a granulocyte count > or = 500 x 10(9)/L in a median of 23, 10, 16, 11, and 11 days, with a platelet count greater than 20,000 x 10(9)/L documented in a median of 22, 23, 32, 12, and 10 days, respectively. The time to granulocyte reconstitution was significantly shorter for patients who received CD34+ PBPCs alone (cohort no. 5), or in combination with CD34+ marrow (cohort no. 4), when compared with those who received only the CD34+ marrow fraction (P < .01). From 1 to greater than 4 logs of breast cancer cell depletion were documented after CD34-selection, for patients in whom tumor was initially detected. CONCLUSION: CD34+ marrow and/or PBPCs provide reliable and timely hematopoietic reconstitution in breast cancer patients receiving high-dose chemotherapy. Contamination of both marrow and PBPCs with breast cancer cells was reduced using this positive selection technique.

Mast cell immunohistochemistry: non-immunological immunostaining mediated by non-specific F(ab')2-mast cell secretory granule interaction.

During investigations of murine and human mast cell immunoreactivity with potential anti-interleukin-4 antibodies, non-specific, non-immunological labelling of mouse and human mast cells became apparent. Non-specific, non-immunological labelling was identified by (i) immunolabelling of mast cells when using control isotype primary antibodies, (ii) ability of conjugated secondary antibodies to label mast cells without prior mast cell exposure to a primary antibody, (iii) extinction of the non-specific labelling and retention of specific labelling when the pH of the diluting and washing buffers is shifted from pH 7.2 to pH 6.0, and (iv) reduction/extinction of the labelling when the antibodies are pre-incubated with soluble heparin prior to immunostaining. The site of the reactivity on the electron microscope level was shown to be confined to the mast cell secretory granules. The results of this study support the hypothesis that non-specific labelling of mast cells results from an ionic interaction between the F(ab')2 segments of antibodies and the heparin constituent of the mast cell secretory granules. This study points out the necessity of stringent controls when using immunohistochemistry to determine mast cell reactivity to various antibodies.

Corticosteroids as immunomodulators.

Corticosteroids are potent anti-inflammatory and anti-immunologic modulators. They interact with cellular receptors, which eventually leads to modification of DNA transcription. These changes sometimes inhibit cell function and sometimes increase production of cell products. A number of effects are seen throughout the organism, leading to downregulation of allergic responses, inhibition of cell-mediated immunity, decreased accumulation of inflammatory cells, and a lessening of proinflammatory stimuli and blood-vessel responses.

Endothelial and fibroblastic activation in scleroderma. The myth of the "uninvolved skin".

We studied the immunohistochemistry of the skin of scleroderma patients to determine the differences (if any) between clinically "affected" and "nonaffected" areas. We examined paired skin biopsy samples from clinically involved forearm skin ("affected") and clinically uninvolved proximal skin ("nonaffected") taken from 19 patients with diffuse scleroderma and from 15 normal control subjects. We stained the sections with antibodies to endothelial leukocyte-adherence molecule type 1 (ELAM-1; to detect endothelial activation) and to procollagen-1 (PC-1; to detect newly formed, unprocessed collagen). There was increased expression of ELAM-1 and PC-1 in sclerodermatous skin as compared with the controls, but there was no difference between clinically affected and nonaffected skin samples. In 10 of 11 patients whose condition was getting worse, endothelial and fibroblast activation preceded fibrosis. Endothelial and fibroblast activation are more widespread in the skin of scleroderma patients than is evident by inspection on physical examination. What appears to be "normal" skin in diffuse scleroderma is already pathologic, as shown by abnormal endothelial activation and procollagen production.

Morphological evidence for chronic mast cell activation after prolonged exposure with supernatants from chronic graft-versus-host splenocytes.

Chronic graft-vs.-host disease (cGVHD) includes a syndrome of inflammatory and fibrotic changes in some respects resembling scleroderma. In the present study we have quantitated the number of peritoneal mast cells (MC) in mice with cGVHD induced across minor histocompatibility barriers. MC were evaluated by staining with toluidine blue. The number of MC decreased significantly (by 25%) at the onset of the cGVHD fibrosis (day 12). Around day 35, MC were virtually undetectable, and started to reappear on day 130. Upon clinical recovery (day 200) a dramatic increase in MC numbers was found (about 8-fold). In addition, we evaluated by electron microscopy the morphology of peritoneal MC, obtained from normal mice and rats, that had been co-cultured on 3T3 fibroblast monolayer in the presence of splenocyte supernatants from mice with cGVHD or from control mice. After 6-8 days of continuous incubation with the cGVHD splenocyte supernatant, MC appeared to be activated, since they displayed an array of heterogenous granules. Few of the granules were dense; many were swollen and pale. Rare granule extrusion was evident. This would indicate that MC underwent a slow activation process due to a factor(s) present in the cGVHD supernatant, different from the classical acute anaphylactic activation.

Dermal mast cell degranulation in systemic sclerosis.

Paired biopsy samples from involved and uninvolved skin were obtained from 19 patients with generalized scleroderma (11 with early, progressive disease and 8 with late, improving disease). Skin biopsy samples were double stained for mast cell granules and for mast cell membrane. The number of mast cells was increased in patients with systemic sclerosis (SSc), in both involved and uninvolved skin and in both early and late disease. There was an increase in the number of degranulated mast cells in the involved skin of patients with both early and late disease and in the not-yet-involved skin of patients with early disease; however, there was no increase in the number of degranulated mast cells in areas of previously involved but now normal skin of patients with late disease. Increases in mast cell number and degranulation precede clinically apparent dermal fibrosis in SSc. These observations and the absence of mast cell degranulation in regressing skin suggest a participatory role of the mast cell in the clinical progression of skin changes in SSc.

Immunoglobulin dysregulation in murine graft-vs-host disease: a hyper-IgE syndrome.

Immunoglobulin production, particularly IgE, is known to be dysregulated in graft-vs-host disease (GVHD). We examined serum levels of the highly T-dependent Ig isotypes, IgE, IgG1, and IgG2a, in two different mouse models of GVHD. GVHD across minor histocompatibility barriers is produced by injection of B10.D2 spleen cells into 600 rad irradiated BALB/c hosts. Both strains are H2d and mls b, but differ at the minor histocompatibility antigens. As GVHD progresses there is a rapid rise in serum IgE (300-fold) and IgG1 (2.5-fold) with a peak at Day 14. Concomitantly, IgG2a falls. Serum immunoglobulin levels return to normal by 11 weeks. The rise in IgE is abolished by increased (900 rad) recipient irradiation, suggesting that host-derived factors are important. GVHD across major histocompatibility barriers is produced by injection of DBA/2 spleen cells into unirradiated or 600 rad irradiated (B6 x DBA/2)F1 hosts. Only in the irradiated recipients is there severe Ig dysregulation. In this situation there is a 100-fold rise in IgE, and 5- to 10-fold rises in IgG1 and IgG2a. While the results in GVHD across minor barriers suggest stimulation of T helper cells secreting IL-4, the increase in IgE, IgG1, and IgG2a levels in GVHD across major barriers suggests activation of IL-4 and IFN-gamma-secreting T cells. These results indicate that different mechanisms may be operating in these two models of GVH. Murine GVHD can serve as a model for studying dysgammaglobulinemias in general and for hyper-IgE formation in particular.

Histamine release from mouse and rat mast cells cultured with supernatants from chronic murine graft-vs-host splenocytes.

There is growing interest in studying pathways of mast cell activation. In a mouse model of chronic graft-vs-host disease (cGVHD) extensive mast cell activation and degranulation occurs in vivo coincident with the development of dermal fibrosis. An interesting feature of this model is that the mast cell reaction is slow to develop, occurring over a period of weeks and waning by 300 days. The aim of our work was to investigate the effects of supernatants from splenocytes of such cGVHD mice (cGVHD sups) on mouse and rat peritoneal mast cells cocultured with 3T3 skin fibroblasts. We found that cGVHD sups are able to release histamine from both mouse and rat cultured mast cells in a slow fashion. Histamine release became evident only after 5-8 days of coculture of the mast cells with the cGVHD supernatants and thereafter decreased to basal levels. Mast cell activation due to cGVHD supernatants was a noncytotoxic event as demonstrated by mast cell counts in the cocultures and by the ability of mast cells to exclude trypan blue. Mast cells that had been activated by incubation with the cGVHD sups were as responsive to stimulation with either anti-IgE antibodies or compound 48/80 as were mast cells incubated with control sups. Supernatants from mice early in GVHD (Days 11-28) were most active in promoting histamine release. Supernatants from spleens of mice which had GVHD for 290 days and where the mast cells had returned to full granulation in vivo were inactive. This is the first in vitro study demonstrating slow mast cell histamine release instituted by other cells, namely the splenocytes of cGVHD mice.

Mast cells and fibrosis. The relevance to scleroderma.

Mast cells are being recognized as important constituents in fibrotic processes. This article reviews the evidence for increased mast cell numbers and/or function in a variety of fibrotic conditions. Increased mast cell numbers and activity are seen in chronic murine graft-versus-host disease (a model for scleroderma) and in active scleroderma itself. An integrated schema for scleroderma is presented, emphasizing the interactions among mast cells, endothelial cells, and fibroblasts mediated by heparin and heparin-binding growth factors.

IL-3, IL-4, and IL-6 enhance IFN-gamma-dependent bone marrow natural suppressor activity.

The ability of murine bone marrow (BM) natural suppressor (NS) cells to suppress a Con A proliferation assay was greatly enhanced by supernatant obtained from the T cell hybridoma D9C1.12.17. Of the lymphokines produced by this hybridoma, three were found to enhance suppression: interleukin-3 (IL-3), IL-4, and IL-6. These molecules enhanced suppression of both unirradiated and irradiated (2000 R) BM cells indicating that augmented suppression was not just due to proliferation of NS cells. The ability of all three of the lymphokines to enhance BM suppression could be blocked by anti-interferon-gamma (IFN-gamma) antibody. These results indicate that (1) NS cell activity is not radiosensitive and (2) that two signals may be required for maximal NS cell suppression, one being a lymphokine-mediated signal and the other IFN-gamma.