Hans-Wilhelm Kreysel

Evidence that defective interferon-gamma production in atopic dermatitis patients is due to intrinsic abnormalities.

The in vitro production of interferon-gamma (IFN-gamma) in 19 atopic dermatitis (AD) patients was compared with that of 12 controls. IFN-gamma production by phytohaemagglutinin (PHA) stimulated peripheral blood mononuclear cells (PBMC) was profoundly diminished in AD patients, whereas the proliferative response was similar to that of control PBMC. The addition of 40 U/ml of interleukin-2 (IL-2) to the cultures failed to restore IFN-gamma production. Similarly, removal of adherent cells also had no effect. Reduced IFN-gamma secretion was observed after stimulation with the CD3 monoclonal antibody OKT3, ionomycin + 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or with high levels of IL-2 (200 U/ml). There were increased proportions of CD4+ T helper/inducer cells and decreased proportions of CD8+ T cytotoxic-/suppressor cells and CD16+ natural killer (NK) cells in AD patients. This resulted in an increased CD4/CD8 ratio as compared with controls, but no correlation was observed between numbers of T cell subpopulations and IFN-gamma generation. However, a significant correlation was found between IFN-gamma generation in vitro and IgE serum concentration in AD patients. The data suggest that the decreased production of IFN-gamma by AD patients is due to intrinsic differences in capacity to produce this cytokine and is not the result of differences in regulatory cell interactions. Moreover, the findings indicate that decreased production of IFN-gamma may be an important factor in the pathogenesis of this disease.

Inflammatory mediators potentiate pain induced by experimental tissue acidosis.

&NA; Electrophysiological evidence from cutaneous nociceptors suggested a synergism between excitatory actions of inflammatory mediators (IM) and low pH. In human skin it is possible to induce constant ongoing pain with continuous infusion of acid buffer. This method was used to study the interaction with mediators of inflammation psychophysiologically. A skin area on the palmar forearm of 6 subjects (either gender, age 22–35 years) was continuously infiltrated with a phosphate buffered electrolyte solution (pH 5.2) using a motorized syringe pump that was adjusted so as to produce constant pain of about 20% on a visual analog scale (VAS; extending from ‘no’ to ‘unbearable pain’). Pain was assessed on the VAS at 10‐sec intervals; the rating was called up by means of an acoustic signal. An additional cannula was placed in the skin before the infusion of acidic buffer started. Injections of an acidic combination of IM (BK, 5‐HT, HIS, PGE2) 0.2 ml were then given through the cannula at intervals of 10 min in a randomized double blind order of concentrations. The other arm was used for negative control, i.e. IM in neutral solution were injected into normal skin continuously infiltrated with a buffer solution at pH 7.4. The IM induced dose‐dependent, transient burning pain on both arms — markedly more intense and prolonged, however, in the acidotic skin (P < 0.004, U‐test). The difference corresponded to a 10‐fold increase in algogenic potency with 10−7 M IM, being smaller with 10−6 and 10−5 M concentration. The interaction between low pH and IM was mutual: additional injections of plain phosphate buffer (pH 5.2) into the acidotic skin were significantly more painful (20‐fold) after application of IM than under control conditions. Thus, we tend to conclude that it is the inflammatory mediators that potentiate the algogenic effect of low pH rather than vice versa. Tissue acidosis appears as a dominant factor in inflammatory pain.

Immunoglobulin E and Immunoglobulin G Subclass Distribution in vivo and Relationship to in vitro Generation of Interferon-Gamma and Neopterin in Patients with Severe Atopic Dermatitis

In vitro interferon-gamma (IFN gamma) and neopterin generation by peripheral blood mononuclear cells (PBMC) from 15 patients with severe atopic dermatitis (AD) and 10 healthy controls was investigated. A significant proportion of patients had an impaired capacity to secrete IFN gamma after phytohemagglutinin (PHA) stimulation in vitro and therefore IFN gamma production was significantly lower compared to controls. Neopterin generation in vitro did not differ significantly from that of controls and no correlation between in vitro IFN gamma and neopterin production could be observed in either group. Analysis of serum IgG subclass distribution showed that patients with AD. had increased IgG4 serum concentrations while IgG1, IgG2 and IgG3 levels did not differ significantly from those of controls. Surface marker analysis revealed increased numbers of CD23+ lymphocytes in patients with AD which was positively correlated with the serum IgG4 and IgE concentration. Furthermore, a significant correlation was found between IFN gamma generation in vitro and IgE and IgG4 concentration in vivo in AD. The data suggest that a possible dysregulation of IFN gamma, interleukin-4 or other lymphokine interleukin-4 or other lymphokine production may be related to increased IgE and IgG4 production and seems to be an important factor in the pathogenesis of AD.

Functional characterization of skin-infiltrating lymphocytes in atopic dermatitis.

Skin‐infiltrating lymphocytes (SIL) were isolated from skin biopsies of patients with hyperimmunoglobulin E (IgE) atopic dermatitis (AD) and expanded in vitro in the presence of IL‐2 in combination with IL‐4. Phenotypic analysis of skin‐derived cells revealed the predominance of CD4+ T helper/inducer phenotype in SIL populations. In 3H‐thymidine incorporation assays, SIL showed proliferation in response to IL‐2, IL‐3, IL‐4, ionomycin (Io)+ 12‐o‐tetradecanoyl‐phorbol‐13‐acetate (TPA) and OKT3 + TPA. OKT4 with and without TPA did not induce proliferation. Tumour necrosis factor alpha (TNF‐α) did not block proliferative responses of SIL to IL‐2 and IL‐4. Cultured SIL showed no cytotoxic activity against K562 and Jurkat target cells. Expanded skin‐derived T cells were tested for their capacity to secrete several cytokines in vitro. SIL secreted significant amounts of IL‐4, GM‐CSF and TNF‐α upon stimulation with mitogens but failed to secrete IFN‐γ. Io in combination with phorbolester induced the secretion of larger amounts of IL‐4, GM‐CSF, TNF‐α and low amounts of IFN‐γ. The data indicate that SIL derived from AD lesions were defective in their capacity to secrete IFN‐γ but were enriched in T cells capable of producing IL‐4 upon stimulation. The results support the possibility of a predominant‘TH2‐like’ cell‐mediated immune response in lesional skin of AD patients.

Progressive increase of CD7- T cells in human blood lymphocytes with ageing.

CD7 is one of the major surface antigens expressed very early during T cell ontogeny. Lack of CD7 expression on mature T cells is regarded as a classical feature of malignant T cells in certain forms of cutaneous T cell lymphoma. Previously, we identified a CD7‐ subset of peripheral blood T lymphocytes in normal human individuals. In this study we determined the portion of CD7‐ T cells in the peripheral blood of healthy volunteers ranging in age from 8 months to 90 years (n= 85) and in cord blood of full‐term infants (n= 14), Furthermore, this CD7‐ subset was characterized in detail by the use of MoAbs and three‐colour flow cytometry. In cord blood no CD7‐ T cells could be detected. After birth, percentage and absolute number of CD7‐ T cells increased with age. Independently of age, most CD7‐ CD3+ cells belonged to the CD4+ subpopulation. Focusing on the latter we could demonstrate the predominance of the CD45RO+CD45RA‐ phenotype in the CD7‐ subset. Furthermore, CD7‐ T cells contained a higher number of cells expressing activation markers and the CD57 antigen, but a reduced number of CD62L+ cells in comparison with CD7+ T cells.

Selective Alterations in Natural Killer Cell Subsets in Patients with Atopic Dermatitis

We examined the immunophenotypic characteristics of natural killer (NK) cell subsets in patients with severe atopic dermatitis (AD), rhinitis allergica (RA) and in healthy controls. Expression of CD16, CD56 and CD57 antigens on peripheral blood lymphocytes was evaluated by simultaneous double immunocytofluorometry. Our results showed significantly lower percentages of cells with CD16, CD56 and CD57 surface antigens in patients with AD. Furthermore subdivision of the AD group into two subgroups, AD1 and AD0 (with and without antigen-specific IgE antibodies against potent inhalative allergens, i.e. mite, grass, rye, birch, cat) revealed that patients of subgroup AD1 showed a more prominent decrease compared to that of subgroup AD0. Moreover, we found a significant negative correlation between the percentage of CD56 + CD16 + NK cells and total IgE levels in serum, which were significantly higher in patients of subgroup AD1 than in AD0. NK cell activity was deficient in patients with AD but there was no difference between both subgroups. These data indicate that considerable heterogeneity in immunologic regulation may exist in patients with AD with regard to their NK cell subsets.

Tumor‐infiltrating lymphocytes isolated from a Ki‐1–positive large cell lymphoma of the skin. Phenotypic characterization and analysis of cytokine secretion

Tumor‐infiltrating lymphocytes (TIL) were obtained from a biopsy of a patient with a Ki‐1–positive large cell lymphoma of the skin. Immunohistologic studies of the large anaplastic tumor cells showed an “aberrant” T “helper/inducer” phenotype (CD30+CD3–CD4+CD8–IL–2R+HLA–DR+). Using a cDNA probe for the constant region of the T‐cell receptor (TCR) beta gene, the cells were identified by their distinct monoclonal rearrangement of T‐cell receptor (TCR)‐beta DNA. Tumor cells isolated from biopsies were cultured in the presence of interleukin‐2 (IL‐2). Outgrowing lymphocytes were cloned, expanded in vitro, and 11 clones were subjected to phenotypic analysis: ten clones showed a predominantly CD4‐positive T “helper/inducer” phenotype whereas one clone expressed CD8 T “cytotoxic/suppressor” antigens. In contrast to the tumor cells, cells of all clones grown in vitro expressed the TCR‐associated CD3 complex. Furthermore, cells from all clones analyzed expressed CD5, CD7, CD45RO (UCHL1), CD11a (LFA‐1), CD25, and HLA‐DR antigens. Cells of two of ten CD4‐positive clones expressed CD45RA (2H4) in addition to UCHL1. T‐cell clones isolated from the tumor and grown in vitro exhibited individual DNA restriction band patterns different from that of a DNA tumor biopsy specimen. Therefore, the authors conclude that these T‐cell clones represent presumably nonmalignant TIL. All clones tested secreted interferon (IFN)‐gamma and tumor necrosis factor (TNF)‐alpha in vitro. Nine of 11 clones were found to secrete additionally IL‐2 and IL‐4 upon stimulation with phytohemagglutinin (PHA) whereas two clones did not secrete detectable amounts of IL‐4. Selective growth of TIL in the presence of IL‐2 opens the possibility to use these cells in adoptive immunotherapy of cutaneous T‐cell lymphoma (CTCL). Cytokines secreted by TIL cells in vitro (IL‐2, IL‐4, IFN‐gamma, TNF‐alpha) may be involved in their antitumorgenic activity. Moreover, these data implicate that CD4‐positive TIL derived from CTCL cannot be grouped into different subsets based on the production of IL‐2, IL‐4, IFN‐gamma, and TNF‐alpha. 68:2155‐2160, 1991.

Anti-Leu 19 monoclonal antibody detects an antigen on autonomic nerves in human skin

The monoclonal antibody anti‐Leu 19 recognizes the NKH‐1 antigen of active natural killer cells. In immunohistological preparations of the skin, the antibody reacts with cutaneous nerves and labels nerve fibers in the region of the sweat glands, the blood vessels and the hair arrector muscles. This staining pattern corresponds to the distribution of the autonomic sympathetic nervous system of the skin. On the other hand, anti‐Leu 7 labels a myelin‐associated glycoprotein which is to be found in the myelin‐sheath of sensory cutaneous nerves. The reactivity of neural structures of the skin can thus be differentiated. Anti‐Leu 7 labels the myelinated sensory nervous system, whereas anti‐Leu 19 seems to constitute a new marker for the autonomic nervous system of the skin.

Two-color flow cytometry analysis in alopecia areata.

Since previous studies have shown that circulating T cytotoxic/suppressor cells and natural killer cells are reduced in alopecia areata (AA), we attempted to find further imbalances in mononuclear cells by double labeling with these and additional monoclonal antibodies. In all, the blood of 35 patients and 44 controls was examined using the combinations anti-Leu 3a/8, Leu 2a/8, Leu 2a/15, Leu 7/15 and Leu 7/11a. We found a highly significant reduction of the subpopulation Leu 2a+8+ (p less than 0.008). The results show that patients with AA have additional abnormalities of the circulating mononuclear cells, whereby the reduced Leu 2a+8+ subpopulation represents a new additional marker.

Expression of Langerhans cell antigens in the hair follicles in alopecia areata

In the normal hair follicle Langerhans cells (LC) are located predominantly in the outer root sheath. The majority of LC was found in the supraseboglandular part of the follicle, while in the infraseboglandular parts only single LC could be demonstrated. Progressive stages of alopecia areata (AA) are characterized by an increase of CD1-positive cells in the hair bulb, and this was considered as an indication for the involvement of dendritic follicular cells in the pathogenesis of AA. While CD1 is suggested as the best immunomorphological marker for LC, besides electron microscopic characterization, monoclonal antibodies against MHC class II antigens and the CD4 antigen characterize different subsets of functional active LC populations. Therefore the expression of LC antigens CD1, CD4, HLA-DR, -DQ, -DP was examined using the avidin-biotin-peroxidase method in situ in interfollicular and follicular epidermis of 40 patients with untreated AA. The results were compared with those of scalp biopsies from normal controls.