Transepidermal induction of contact hypersensitivity in mice with a water-soluble hapten.

An experimental analysis has been conducted on the capacity of a highly-reactive, water soluble hapten, trinitrobenzene sulfonic acid, to induce contact hypersensitivity when applied epicutaneously to body wall skin of normal mice. Application of plastic chambers containing the hapten to murine skin for as little as 1 h produced readily detectable sensitization in several genetically disparate inbred strains. Moreover, the efficiency of sensitization was found to be similar to that following epicutaneous application of this hapten's lipid-soluble cogener, trinitrochlorobenzene. Using this approach, it has been determined that UVB radiation, intradermally injected tumor necrosis factor-alpha, and epicutaneously applied cis-urocanic acid can impair contact hypersensitivity induction by transepidermally delivered trinitrobenzene sulfonic acid, and that animals that fail to become sensitized proceed to acquire hapten-specific unresponsiveness. It is concluded that epicutaneous sensitization to chemically reactive, water-soluble molecules is experimentally attainable if precautions are taken to insure that contact between the hapten solution and the cutaneous surface is maintained for at least 1 h. Understanding the cellular and molecular mechanisms of sensitization and tolerance induction by epicutaneously applied water soluble haptens may prove to be important in understanding the pathogenesis of allergic contact dermatitis that develops to chemicals in the industrial setting, in the environment, and in the clinic.

Internalization of Ia molecules into Birbeck granule-like structures in murine dendritic cells.

Dendritic cells isolated from the draining lymph nodes of mice sensitized epicutaneously with hapten are potent antigen-presenting cells and contain Birbeck granules and cored tubules characteristic of antigen-activated epidermal Langerhans cells. We used immunogold labeling and transmission electron microscopy to follow the internalization of Ia molecules in these antigen-presenting cells. We found that Ia molecules were internalized into Birbeck granule-like structures in the antigen-activated dendritic cells. Computer reconstruction of serial sections of the dendritic cells demonstrated that these structures span the cytoplasm from the cell membrane to the nuclear membrane and are associated with lysosomes. The internalization of Ia molecules into these structures supports the hypothesis that the Birbeck granule-like structures are derived from the cell membrane and are involved in the antigen-processing/presenting function of the dendritic cells.

Evidence that cutaneous antigen-presenting cells migrate to regional lymph nodes during contact sensitization.

These studies address the hypothesis that Ag-bearing epidermal Langerhans cells migrate to the regional lymph node during contact sensitization and function as APC. Skin from C3H mice was grafted onto BALB/c nude mice, and 7 or 14 days later, the recipients were sensitized with FITC through the grafts. APC from lymph nodes draining the site of sensitization were capable of sensitizing C3H recipients to FITC. Because sensitization is MHC restricted, only cells reaching the lymph node from the grafted skin could have induced contact hypersensitivity in C3H mice. Examination of the FITC+ draining lymph node cells by immunofluorescence and immunoelectron microscopy demonstrated that all were Ia+, most were F4/80+, and some contained Birbeck granules. These studies demonstrate that Ia+, FITC+ cells from the skin, at least some of which are Langerhans cells, leave the skin after epicutaneous sensitization with FITC and participate in the initiation of the contact hypersensitivity response within the regional lymph node.

Comparison of the immunosuppressive effects of cyclosporine, lipid-soluble anesthetics, and calmodulin antagonists. Response to exogenous interleukin 2.

The purpose of these investigations was to compare the immunosuppressive mechanism of cyclosporine (CsA) with those of lipid-soluble local anesthetics and calmodulin antagonists. Chlorpromazine (CPZ) and pentobarbital (PB) both inhibit lymphocyte activation by attenuating sodium and potassium ion potentials. CPZ and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) can also block calcium-dependent activation processes by inhibition of calmodulin and protein kinase C. All four compounds were found to suppress human and murine lymphoproliferation to both alloantigen or mitogen in a dose-dependent and saturable manner. Exogenous interleukin-2 (IL-2) restored mitogenic responsiveness to cultures suppressed using W-7 and CsA, but not to lymphocytes suppressed with either CPZ or PB. Cytofluorographic analysis revealed that the degree of suppression in drug-treated lymphocytes was significantly correlated with the surface expression of receptors for transferrin and interleukin-2. Inhibition of IL-2 activation by PB was demonstrated to result from a blockade of the mitogenic growth factor signal using the IL-2-dependent cell line HT-2. Thus, the mechanism of action of cyclosporine can be differentiated from those of anesthetic immunosuppressants at the level of responsiveness to interleukin-2. The data support the hypothesis that cyclosporine may be an antagonist of calmodulin that selectively blocks early events in T lymphocyte activation leading to IL-2 synthesis, but does not inhibit the expression or function of the IL-2 receptor.

Elevated adenosine deaminase and purine nucleoside phosphorylase activity in peripheral blood null lymphocytes from patients with acquired immune deficiency syndrome.

The purine metabolic enzymes adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) are important in lymphocyte differentiation, and genetic deficiencies of either enzyme have been associated with hereditary immunodeficiency states. Both ADA and PNP activity were measured in null cell-enriched and T cell-enriched peripheral blood lymphocytes from 16 patients with the acquired immune deficiency syndrome (AIDS), seven patients with the AIDS-related symptom complex (ARC), and seven asymptomatic homosexuals. ADA activity in nmol/10(6) lymphocytes/h was significantly elevated in null lymphocytes from AIDS (161 +/- 12) as compared with 23 healthy heterosexual controls (127 +/- 8;P less than .025). PNP activity was also significantly increased in null lymphocytes from AIDS patients (96 +/- 10;P less than .005) as well as those from ARC patients (84 +/- 11:P less than .025) relative to controls (61 +/- 5). No significant differences in enzyme activity were noted in T cell-enriched cells in any group. Along with elevated enzyme activity, AIDS patients had small yet significant increases in the percentages of HLA-DR (P less than .025), terminal deoxynucleotidyl transferase (TdT) (P less than .0001), and peanut agglutinin receptor (P less than .0001) positive lymphocytes in the null fraction compared with controls. TdT-positive cells appeared morphologically as large lymphoblasts with irregular nuclei. The data imply that the cellular immune deficiency in AIDS is not a result of deficiencies in lymphocyte ADA or PNP activity, but is more likely associated with an increase in an immature and/or activated lymphocyte subset.

Abnormal lymphocyte response to exogenous interleukin-2 in homosexuals with acquired immune deficiency syndrome (AIDS) and AIDS related complex (ARC).

Interleukin-2 (IL-2) production as well as the response of lymphocytes to exogenous IL-2 was measured in asymptomatic homosexuals, patients with acquired immune deficiency syndrome (AIDS), and homosexuals with prodromal symptoms. IL-2 production following lectin stimulation was not significantly different in homosexuals compared to age matched heterosexual controls. In contrast, the response of lymphocytes to exogenous IL-2 was significantly decreased in AIDS and homosexuals with prodromal symptoms compared to asymptomatic homosexuals and heterosexual controls. These data, combined with the data of others, suggest that an underlying immune defect in AIDS is abnormal IL-2 receptor expression and resultant poor IL-2 response.

In vitro modulation of purine enzyme metabolism and lymphocyte surface marker expression by thymosin fraction 5 in homosexual males.

Thymosin fraction 5 (Thymosin) has numerous immunoregulatory activities including modulation of enzymes involved in lymphocyte maturation. The effect of Thymosin on the purine metabolic enzymes adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and 5' nucleotidase (5'NT) in null and T-enriched peripheral blood lymphocytes from sexually active asymptomatic homosexual males (AS), patients with the AIDS-related symptom complex (ARC), and those with acquired immune deficiency syndrome (AIDS) was examined and compared to its effect on lymphocytes from healthy heterosexual controls. Mean ADA activity was significantly higher in null cells from fourteen AIDS patients than in five asymptomatic homosexuals, ten ARC patients, or 27 controls. Mean PNP activity was significantly elevated in null-enriched lymphocytes from ten ARC and fourteen AIDS patients compared to controls. No differences in these enzymes were found in T-enriched cells from any group. 5'NT was markedly decreased in both null and T lymphocytes in all homosexual groups relative to controls. Homosexuals had significantly elevated percentages of OKT10 positive and Ia positive lymphocytes compared to controls. Thymosin at an optimal concentration of 150 micrograms/ml caused significant decreases in mean ADA and PNP activity in null lymphocytes from ARC + AIDS patients along with a significant decrease in the percentage of OKT10 positive lymphocytes. No phenotypic changes were seen in AS or control lymphocytes. The data suggest that Thymosin has a maturational effect in vitro on immature T cells from symptomatic homosexuals.

IL-2 production and response in vitro by the leukocytes of patients with acquired immune deficiency syndrome.

In order to investigate the nature of the T cell defect associated with the acquired immune deficiency syndrome (AIDS) we studied the ability of peripheral blood mononuclear cells from 8 patients with Kaposi's sarcoma (KS), 2 with opportunistic infection (OI), 23 with AIDS-related symptoms complex (ARC) without KS or OI (ARC), and 29 heterosexual controls to produce interleukin II (IL-2) on phytohemagglutinin (PHA) stimulation and to respond to exogenously supplied IL-2. Patients with AIDS as well as those with ARC produced adequate levels of IL-2 in response to lectin stimulation when compared to controls (AIDS, 3.07 +/- 1.98 units; ARC, 3.03 +/- 1.89 units; controls, 3.75 +/- 1.52 units). However, the ability of these patients' cells to respond in vitro to exogenously supplied IL-2 as measured on short-term PHA-stimulated T cell blasts, was found to be severely impaired in patients with AIDS and ARC (AIDS, 22.4 +/- 6.0 X 10(-3) cpm; ARC, 20.1 +/- 4.2 X 10(-3) cpm; control, 41.4 +/- 4.2 X 10(-3) cpm). This impairment was associated with diminished expression of the IL-2 receptor on 7-day-old lectin-stimulated T cells from both patient groups (AIDS, 17.7 +/- 5.7; ARC, 36.8 +/- 4.4; control 71.8 +/- 1.7). These results should be considered when IL-2 is proposed as potential therapy in the treatment of AIDS. They also suggest that the nature of the AIDS defect is related to impaired hormone receptor expression.

Decreased 5' nucleotidase activity in lymphocytes from asymptomatic sexually active homosexual men and patients with the acquired immune deficiency syndrome.

5' Nucleotidase (5'NT) is an ectoenzyme associated with the plasma membrane of most mammalian cells. Low 5'NT activity has been observed in peripheral blood lymphocytes from patients with immunodeficiency states. 5'NT activity was measured in null and T-enriched lymphocytes from asymptomatic homosexual men and from 20 men with various degrees of the acquired immune deficiency syndrome (AIDS). Asymptomatic homosexuals were self-referred because of their concern about AIDS and were not necessarily representative of homosexuals in the general population. Enzyme activity was significantly decreased in both null (7.0 +/- 2.4 nmol/10(6) cells/h) and T-enriched (12.0 +/- 6.0 nmol/10(6) cells/h) lymphocytes in homosexuals as compared to lymphocytes from aged-matched heterosexual male and female controls (null = 10.8 +/- 6.5 and T = 22.3 +/- 10.6, P less than .0001 and .008, respectively). Decreased activity was present regardless of whether the patients were asymptomatic, had prodromal symptoms such as fever, lymph node enlargement, weight loss and diarrhea, or had opportunistic infections or Kaposi's sarcoma. Homosexuals had a significantly higher fraction of lymphocytes expressing the activation antigens T10 (20% +/- 3.3%) and Ia (13% +/- 2.9%) than controls (11% +/- 1.8% and 5% +/- 0.8%, respectively, P less than .05). They also had a significantly lower fraction of OKT4-positive helper lymphocytes than controls (22% +/- 3.4% v 35% +/- 2.2%, P less than .05). 5'NT activity in lymphocytes enriched for null cells from homosexuals correlated inversely with the percentage of Ia-positive lymphocytes (r = -.655; P less than .02). There was no correlation between 5'NT activity and the percentage of T4- or T8-positive lymphocytes or the T4/T8 ratio. Moreover, 5'NT activity was significantly decreased in both OKT4 (P less than .025) and OKT8 (P less than .05) enriched lymphocytes in homosexuals compared to controls. The data suggest that decreases in 5'NT may be a generalized defect of the peripheral blood T lymphocytes from active homosexuals that is independent of increases or decreases in specific T subpopulations or clinical status. It may contribute to the pathogenesis of AIDS.

T cell surface antigen expression on lymphocytes of patients with AIDS during in vitro mitogen stimulation.

Surface marker expression on peripheral blood mononuclear cells (PBMC) was evaluated daily in PHA- and PWM-stimulated cultures of eight AIDS patients and eight normals. Before culture, the patients' cells showed the characteristic decrease in OKT 4+ cells (normals 40.4%, patients 22.3%; P less than 0.001), increase in OKT 8+ cells (normals 27.6%, AIDS 38.4%; P = 0.002), increase in OKT 10+ cells (normals 15.5%, AIDS 42.8%; P = 0.002), and increase in HLA-DR+ cells (normals 11.4%, AIDS 28.7%; P = 0.01). The percentage of OKT 11+ cells remained unchanged, while the percentage of OKT 3+ cells dropped over the first 2 days in PHA but not in PWM cultures of both groups (PHA: normals 69.8% to 35.1%; P = 0.001, AIDS 56.5 to 38.5%; P = 0.001, PWM: normals 62.8%-65.9%, AIDS 66.8% to 63.9%), and recovered in both groups by day 5. In PWM cultures OKT 3+ cells increased significantly in normals but not in AIDS (normals 62.6%-77.7%; P = 0.04, AIDS 61.8 to 48.7%). OKT 4 expression decreased in normal PHA cultures after 1 day (38.9% to 29.6%; P = 0.05) and then recovered by day 5. Its expression increased in AIDS PHA cultures by day 5 (18.0%-41.1%; P less than 0.001). The final percentage of OKT 4+ cells in AIDS cultures was within the normal range (35.0%-49.0%). OKT 8 expression increased in both study groups after PHA stimulation (normals 29.5%-50.4%; P = 0.002, AIDS 37.4%-50.7%; P = 0.02) and in normals but not AIDS after PWM stimulation (normals 28.9%-35.5%; P = 0.004, AIDS 38.5%-35.6%). Because of the relative changes in expression of OKT 4 and OKT 8, the 4/8 ratio declined in the normal PHA cultures (1.89 to 1.03; P = 0.1) and increased in the AIDS cultures (0.68-1.18; P = 0.09). Also, the sum of OKT 4+ and OKT 8+ cells in PHA cultures increased from 68% to 94% whilst expression of OKT 11 remained unchanged, indicating co-expression of these antigens on individual cells. Both PHA-and PWM-stimulated normal cells showed an increase in OKT 10 (PHA 16.0%-53.4%; P = 0.01, PWM 16.1%-33.9%; P = 0.03) and HLA-DR (PHA 8.6%-27.3%; P = 0.03, PWM 12.5%-26.6%; P = 0.07). In AIDS PHA cultures this did not change, and in their PWM cultures OKT 10 expression declined (44.8 to 23.0%; P = 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Impaired T-cell mitogen responses in some patients with thalassemia intermedia.

Ten thalassemia intermedia patients were tested for their mitogenic responses to phytohemagglutinin P (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM). Impaired responses to PHA and Con A were observed in patients with serum-iron levels higher than 200 microgram/dl. Responses within or slightly higher than those in the normal range were observed in patients with serum-iron lower than 200 microgram/dl. By contrast, no significant changes occurred in the majority of the responses to PWM.