Soluble serum interleukin 2 receptors in patients with systemic sclerosis.

OBJECTIVE: To determine whether soluble interleukin 2 receptor (sIL-2R) levels correlate with natural history of disease in patients with systemic sclerosis (SSc). METHODS: The following groups were studied: Group I included 81 consecutive new patients with SSc. Group II consisted of 21 patients with diffuse cutaneous (dc) SSc whose peripheral blood and affected skin had been analyzed for T lymphocyte subsets. Group III contained 38 patients with dcSSc with serial sIL-2R determinations during the course of disease. sIL-2R was performed using a commercial double monoclonal antibody ELISA technique. RESULTS: The 81 Group I patients with SSc had a mean sIL-2R level of 821 units compared with 35 controls, who had a mean of 389 units (p < 0.001). sIL-2R level significantly correlated with the extent of skin thickening (p < 0.005). In Group II patients, sIL-2R was found to correlate with the CD4 to CD8 ratio. Blindly assessed clinical evidence of disease activity from the serial samples of 38 Group III patients was consistent with sIL-2R levels in 83% of the samples. There was high correlation of change in sIL-2R with change in skin score over time in Group III subjects (r = 0.71). CONCLUSIONS: These results suggest a role for T cell activation in the pathogenesis of SSc. sIL-2R levels may be useful adjunct to clinical evaluation in assessing disease activity and predicting future events in patients with SSc.

Cutaneous T-cell lymphoma: utility of antibodies to the variable regions of the human T-cell antigen receptor.

BACKGROUND: The clonotypic 90 kd Ti heterodimer of the human T-cell antigen receptor is composed of two distinct chains (alpha beta or rarely tau delta) that result from the recombination of variable (V), constant, joining, and, in the case of beta chains, additional diversity regions. OBJECTIVE: The variable region expression of human cutaneous T-cell lymphoma (CTCL) was studied. METHODS: Biopsy specimens from 13 patients with CTCL (7 plaque, 3 tumor stage, 3 Sézary syndrome) were examined immunohistochemically by a panel of seven commercially available monoclonal V-region antibodies. RESULTS: Two patients had significant anti-V-region staining. One patient with Sézary syndrome had two lesions, subjected to biopsy 4 months apart, that reacted with beta V5(a), a specificity previously documented by flow cytometry of leukemic cells. A patient with plaque-stage CTCL, negative for T-cell gene rearrangement by Southern blot, demonstrated reactivity with beta V5(c) largely limited to epidermotropic lymphocytes. CONCLUSION: Panels of V-region antibodies should be useful reagents for diagnosis and follow-up of CTCL.

A monoclonal antibody recognizing Golgi apparatus produced using affinity purified material from a patient with connective tissue disease.

Serum antibodies recognizing the Golgi apparatus have been reported in patients with connective tissue diseases, but little is known of their significance. Serum from a systemic lupus erythematosus patient with polymyositis was found to have high titers of anti-Golgi apparatus antibody. This serum recognized a 64 kD polypeptide in immunoblotting with HEp-2 cells. To verify that the 64 kD polypeptide was associated with the Golgi apparatus and to characterize which Golgi component was recognized, a monoclonal antibody was produced. IgG, isolated from this serum, was used in affinity chromatography to produce purified material which was used to generate a mouse monoclonal antibody. The monoclonal antibody had an indirect immunofluorescent pattern identical to that produced by the patient's serum, and similarly recognized a 64kD polypeptide in immunoblotting. A 59 kD polypeptide was also recognized by the monoclonal antibody, suggesting that the antigens recognized by the monoclonal and serum antibodies may be only partially identical. The antigen appears to be a glycoprotein and an integral component of the Golgi cisternae membranes.

Establishment of a human cutaneous T-cell lymphoma in C.B-17 SCID mice.

Investigation into the immunobiology of cutaneous T-cell lymphomas (CTCL) would be facilitated by the development of a suitable experimental system. The recent use of mice with severe combined immune deficiency (SCID) as a vehicle to study the human immune system prompted us to try to establish CTCL in SCID mice. We found that a CD4+ lymphocytic infiltrate characteristic of CTCL was maintained within patient skin grafts in place on natural killer cell depleted SCID mice for the month of observation. CTCL cells were not found outside the human skin graft. This chimeric model using SCID mice and patient lesional skin should provide a useful tool to characterize CTCL/skin microenvironmental interactions and to test new therapeutic approaches.

Studies of connective tissue mast cell-mediated cytotoxicity.

Although mast cells have been implicated in mediating antitumor activity, the kinetics, mechanism(s), and suspectibility of different tumors to mast cell-mediated cytotoxicity have not been defined. Rat connective tissue mast cells (CTMC) of greater than or equal to 99% purity were investigated in vitro and found to express maximal spontaneous cytotoxicity against the mouse fibrosarcoma cell line WEHI-164 (56.0% +/- 2.1 SEM), the ultraviolet B (UVB)-induced, cutaneous fibrosarcoma 5C25 (34.7% +/- 3.4 SEM), and the human renal cell tumor Currie (26.8% +/- 2.0 SEM) at an effector to target (E:T) ratio of 80:1. Kinetic studies of CTMC-mediated cytotoxicity demonstrated significant detectable lysis against these tumors within 8 h, which was maximal by 16 h. Binding experiments showed that CTMC formed conjugates with all three lytic-sensitive targets; however, CTMC also attached to the lytic-resistant target YAC-1, indicating that conjugate formation alone is not sufficient for mast cell-mediated cytotoxicity. At two different concentrations, mast cell granules (MCG) lysed WEHI-164 (36.5% +/- 6.8 SEM) and 5C25 (34.4% +/- 6.9 SEM), but were only slightly cytotoxic (5.7% +/- 2.9 SEM) against Currie. A potential role for tumor necrosis factor-alpha (TNF-alpha) in CTMC-mediated cytotoxicity also was investigated. Polyclonal antibodies to TNF-alpha greatly reduced CTMC and TNF-mediated lysis of WEHI-164, but only partially inhibited CTMC killing of the slightly TNF-sensitive 5C25 tumors, and had no effect on CTMC cytolysis of Currie. Thus, this study demonstrates that CTMC mediate cytotoxicity in vitro by both TNF-associated and TNF-independent mechanisms. We conclude that CTMC are capable of mediating antitumor activity and that this effect may be important for tumor surveillance in the skin and other sites.

Mechanism of anti-asialo GM1 prevention of graft-vs-host disease: identification of allo-antigen activated T cells.

The purpose of this study was to begin to dissect the mechanism whereby anti-asialo GM1 (alpha ASGM1) prevents otherwise lethal graft-vs-host disease (GVHD) across multiple minor histocompatibility barriers in mice. Phenotypic characterization of cells from the peak proliferative time of the graft-vs-host reaction (C57BL/6J lymph node cells----irradiated LP/J, days 5-7) revealed the alpha ASGM1 and alpha Thy 1.2 identified cells with an approximate 80% concordance and that NK-1.1 staining was negligible. Because resting T cells do not label with alpha ASGM1, the epitope recognized by alpha ASGM1 on GVHR T cells is an activation antigen. Because asialo GM1 has been previously found on the surface of activated macrophages, we wanted to distinguish between the two most likely targets for the in vivo effect of alpha ASGM1 infusions (T cells or macrophages). We compared the effects of alpha ASGM1 infusions on alloantigen-stimulated T-cell proliferation versus antigen presentation: T-cell proliferation was markedly reduced by alpha ASGM1 infusions, whereas antigen presentation function was not diminished. We conclude that the mechanism whereby alpha ASGM1 prevents GVHD does not involve NK cells or antigen presenting cells, but does involve activated donor T cells. The potential therapeutic advantage of such an antibody for use in human disorders compared to pan-immunosuppression lies in its ability to eliminate selectively those T cells involved in the immunologic process (i.e. activated T cells) while sparing the remainder of the T-cell repertoire.

Basic science conferences in residency training: a national survey.

Basic science teaching is an important component of dermatology residency training, and the basic science conference is the major tool utilized by departments of dermatology for its implementation. To characterize the role of basic science conferences in dermatology training, a national survey of chief residents was conducted. Although the survey confirmed that a high value is placed on basic science conferences, a surprising finding was a significant level of dissatisfaction among chief residents, particularly those from university-based programs. Results of the survey have been used to redefine our own objectives in basic science teaching and to propose elements of methodology and curriculum.

Lethal graft-vs-host disease across major histocompatibility barriers: requirement for leucyl-leucine methyl ester sensitive cytotoxic T cells.

L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) is selectively toxic for human natural killer (NK) cells and cytotoxic T lymphocytes (CTL) at both the precursor and effector stage of differentiation. The present studies explored the effects of Leu-Leu-OMe on murine spleen cell function. Leu-Leu-OMe exposure removed NK function from murine spleen cells but spared their capacity to proliferate in response to lipopolysaccharide and Con A. The capacity to generate CTL from both L3T4 (+) and Lyt-2 (+) precursors was lost after Leu-Leu-OMe treatment, whereas alloantigen-induced proliferation and interleukin 2 (IL 2) production by L3T4 (+) T helper cells remained intact. Lethal graft vs host disease (GVHD), which developed in irradiated (C57BL/6 X DBA/2)F1 recipients of C57BL/6 bone marrow and spleen cells was completely prevented by Leu-Leu-OMe treatment of donor cells. In contrast depletion of Lyt-2 positive cells from the donor inoculum did not prevent acute GVHD in this fully major histo-compatibility complex (MHC) incompatible strain combination. However, Leu-Leu-OMe treatment of the Lyt-2 depleted inoculum completely prevented lethal GVHD, although the treated cells retained the capacity to proliferate and secrete IL 2 normally after in vitro stimulation with (C57BL/6 X DFA/2)F1 spleen cells. These findings indicate that L3T4 (+) T helper cells alone are unable to initiate lethal GVHD in this H-2 incompatible strain combination. Rather, lethal GVHD requires the transfer of a Leu-Leu-OMe sensitive T cell subset, likely to be thymus educated pre-CTL. Leu-Leu-OMe treatment should provide a useful way to delineate subpopulations of cells involved in the production of lethal GVHD and an approach to preventing this complication of bone marrow transplantation.

Studies addressing the mechanism of anti-asialo GM1 prevention of graft-versus-host disease due to minor histocompatibility antigenic differences.

In mice, as in humans, lethal graft-versus-host disease (GVHD) with skin involvement often occurs in immunoincompetent recipients of donor hematopoietic cells in spite of matching at major histocompatibility loci and nonreactivity in mixed lymphocyte culture, if donor and recipient are disparate at several minor histocompatibility loci. In mice, both death and skin disease can be prevented by the use of an antiserum containing antibodies to a cell surface glycolipid, asialo GM1 (ASGM1). Because treatment of only the recipients with anti-asialo GM1 substantially reduces the subsequent proliferation of infused donor lymphoid cells, we infer that anti-asialo GM1 interferes with a host minor-antigen-presenting cell, so that donor lymphocytes fail to see minor host antigens as immunogenic. Of the tissues examined by immunofluorescence microscopy, ASGM1 was found on the epidermal Thy-1+ dendritic cell, on dendritic cells in the thymus, and as has been previously described, on lung and spleen cells. Following the intravenous administration of anti-asialo GM1, only the spleen showed an obvious change, losing approximately 80% of its ASGM1 + cells. Further analysis of spleen cells bearing ASGM1 may better define the phenotype of the inferred minor antigen-presenting cell and lead to a method of improving the outcome of human bone marrow transplantation.

Cutaneous mast cell maturation does not depend on an intact bone marrow microenvironment.

We sought to determine whether the maturation of murine cutaneous mast cells from stem cells depends on an intact bone marrow microenvironment. Normal bone marrow cells (+/+) were infused into 2 groups of mast cell-deficient mice: WBB6F1-W/Wv mice and 89Sr-pretreated W/Wv mice. 89Sr is a long-lived bone-seeking radioisotope which provides continuous irradiation of the marrow and thereby ablates the marrow microenvironment. Skin biopsies revealed that the 89Sr-pretreated mice and the controls had repopulated their skin with mast cells equally well. Natural killer cell function was significantly depressed in the 89Sr-treated mice, confirming that the marrow microenvironment had been functionally altered. We conclude that, although the precursors for cutaneous mast cells are marrow derived, they do not need an intact marrow microenvironment for maturation.

Murine graft-versus-host skin disease: a chronologic and quantitative analysis of two histologic patterns.

Human graft-versus-host disease (GVHD) has several cutaneous manifestations, including a lichenoid and a sclerotic injury pattern. A versatile animal model of graft-versus-host skin disease (GVHSD) would facilitate study of the pathophysiology of these two cutaneous injury patterns. We have examined two murine chimeras histologically and have found two distinct patterns. Allogeneically transplanted B1/6 mice show a prolonged lichenoid-interface dermatitis that eventuates in clinical alopecia, whereas LP/J recipients of allogeneic cells do not show hair loss. Their histopathology consists of an early lichenoid phase that abates and is replaced by dermal sclerosis. Because of the versatility of the mouse as a laboratory animal, we feel that this model provides an excellent opportunity to define the immunopathologic mechanisms responsible for skin injury in GVHD. In addition, an understanding of the pathogenesis of the T cell-dependent, lichenoid, and sclerotic patterns of tissue injury in GVHSD might well provide insight into the pathogenesis of the GVHSD analogs, cutaneous lupus erythematosus and scleroderma.

Prevention of lethal, minor-determinate graft-host disease in mice by the in vivo administration of anti-asialo GM1.

Graft-vs-Host disease (GVHD) remains a devastating problem in human bone marrow transplantation (1, 2). Because removal of Thy-bearing cells from the donor inoculum has prevented GVHD in murine models (3, 4), it has been hoped that a similar cell surface antigen or combination of antigens could be found in humans. Unfortunately, treatment of human donor cells with various T cell antisera has not yet been successful in preventing GVHD (5). Encouraging results have been reported in five patients who received bone marrow depleted of T cells by the sequential use of soybean agglutinin and the differential sedimentation of cells forming rosettes with sheep red blood cells (6). Although donor T cells are thought to be necessary for initiating GVHD, the immunopathogenesis of GVHD is still not understood. Because donors and recipients are routinely major histocompatibility complex matched and chosen to be nonreactive in mixed lymphocyte cultures human GVHD is thought to result from minor histocompatibility antigen disparities. Lopez and coworkers (7, 8) found a strong association between the incidence of human GVHD and the pretransplant levels of natural killer (NK) activity of the recipients; when the recipient NK activity was low, GVHD rarely developed. They speculated that the NK cell lineage is serving as an important stimulator-inducer. We therefore examined the in vivo effects of anti-asialo GM1 on a murine model of GVHD based on minor antigen disparity. This antiserum has several immunologic effects, including a profound NK suppression. We found that the mice treated with this antibody have normal survival rates, even though they do develop histologic GVHD in the skin. This finding suggests the possibility of a new prophylactic approach to human GVHD and raises many questions regarding the function of asialo GM1-bearing cells in immune regulation.

Clearing of subacute cutaneous lupus erythematosus around molluscum contagiosum lesions.

A patient with widespread subacute cutaneous lupus erythematosus (SCLE) developed halos of uninflamed, normal-appearing skin around lesions of molluscum contagiosum. A biopsy confirmed the lack of active lupus erythematosus (LE) in the halo area (neither basal layer liquefaction nor inflammatory infiltrate was present). Direct immunofluorescence of the halo area showed only weak staining with IgG and C3. In contrast, adjacent skin, clinically involved with cutaneous LE, showed both active LE by light microscopy and strong staining with IgG and C3. We feel the molluscum contagiosum lesions that developed in areas of active SCLE somehow suppressed the clinical, histologic, and immunologic expression of SCLE in he surrounding skin. This observation might offer some insight into the poorly understood pathophysiologic mechanisms involved in cutaneous LE.