Dick M. Boorsma

Differential expression of CXCR3 targeting chemokines CXCL10, CXCL9, and CXCL11 in different types of skin inflammation

Recruitment of activated T‐cells to the skin is a common feature in a wide variety of inflammatory skin diseases. As CXCR3 activating chemokines CXCL10 (IP‐10), CXCL9 (Mig), and CXCL11 (IP‐9/I‐TAC) specifically attract activated T‐cells, this study addressed the question of whether differences in the expression of these chemokines correlate with the site and cellular composition of the skin infiltrates in different types of inflammatory skin disease. Skin biopsies from lichen planus, chronic discoid lupus erythematosus, allergic patch test reactions, psoriasis, and Jessners lymphocytic infiltration of the skin were investigated for chemokine expression using RNA in situ hybridization, and for the expression of CXCR3 using immunohistochemistry. The results showed differential expression of CXCL10, CXCL9, and CXCL11, which correlated with differences in the localization and cellular composition of the infiltrates. Whereas CXCL10 and CXCL11 were mainly expressed by basal keratinoctyes, CXCL9 mRNA expression was located predominantly in the dermal infiltrates. Correlation with immunohistochemical data suggested that macrophages and activated keratinocytes were the main producers of these chemokines. CXCR3 was expressed by a majority of both CD4+ and CD8+ infiltrating T‐cells, suggesting a functional interaction between locally produced chemokines and CXCR3‐expressing T‐cells. In conclusion, these findings indicate that these CXCR3 activating chemokines play a significant role in the recruitment and maintenance of T‐cell infiltrates in the inflammatory skin diseases studied. Copyright

The antipsoriatic drug dimethylfumarate strongly suppresses chemokine production in human keratinocytes and peripheral blood mononuclear cells.

Background The effectiveness of systemic treatment of psoriasis with fumaric acid esters has been proven, but their mode of action at the cellular and molecular level has not yet been fully elucidated.

Three Epitope-Specific Monoclonal Antibodies against the Hapten Penicillin

Three monoclonal antibodies (Pen 4, Pen 7 and Pen 9) were raised against the benzylpenicilloyl group and used to investigate the antigenicity of this group. The binding of Pen 4 to carrier-bound penicillin derivatives was shown in an ELISA to be dependent on the structure of the side chain in the derivative. Hence Pen 4 recognizes this side chain. From the difference in binding to carrier-bound penicillin derivatives in a competitive enzyme immunoassay it was concluded that Pen 7 mainly recognizes the new antigenic determinant which emerges from the binding of the penicillin derivative to a carrier. The binding of Pen 9 to carrier-bound penicillin derivatives was not influenced by the nature of the side chain. Neither was the bound or free nature of the derivative of influence on the binding. Therefore it is concluded that Pen 9 mainly recognizes the thiazolidine ring of penicillin. This study thus shows that in the benzylpenicilloyl group at least three epitopes can be recognized.

Chemokine IP-10 expression in cultured human keratinocytes

Abstract IP-10, a member of the CXC family of chemokines, is considered to play an important role in inflammation via its T-cell chemotactic and adhesion-promoting properties. Elevated IP-10 levels in the epidermis of psoriasis, delayed-type hypersensitivity reactions, cutaneous T-cell lymphoma and fixed drug eruptions prompted us to study its expression in keratinocytes. IP-10 mRNA could be detected using the sensitive RT-PCR method, but not by Northern blotting in RNA preparations from unstimulated normal cultured keratinocytes, indicating a low steady-state level of IP-10 mRNA. Upon stimulation with IFN-γ, IP-10 mRNA was found to accumulate in high amounts in a time- and dose-dependent manner. Superexpression was found with the combination of IFN-γ and TNF-α or IL-1, although these latter cytokines by themselves did not induce accumulation of IP-10 mRNA. Nuclear run-on experiments performed to investigate the regulation of IP-10 mRNA expression, showed a very high constitutive transcriptional activity of the IP-10 gene in unstimulated keratinocytes, which was not affected by stimulation with IFN-γ, TNF-α, or a combination of IFN-γ and TNF-α. Protein kinase C (PKC) was shown to be involved in IP-10 mRNA expression since the PKC inhibitor H7 decreased IP-10 mRNA accumulation. A protein was isolated from culture supernatants of stimulated keratinocytes using HPLC techniques and, by sequence analysis, was found to be identical to IP-10. The dynamics of secretion of IP-10 protein as monitored by ELISA was shown to parallel the mRNA expression.

Increased CCL27-CCR10 expression in allergic contact dermatitis: implications for local skin memory

Allergic contact dermatitis (ACD) is a T‐cell‐mediated disease in which expression of a distinct repertoire of chemokines results in the recruitment of effector T cells into the skin. While it is becoming clear which chemokines and receptors determine the development of ACD, the mechanisms involved in the retention of T cells in the skin after resolution of inflammation are still unknown. Unravelling these mechanisms will help us to understand local skin memory as observed in retest reactivity and flare‐up reactions. This study was designed to evaluate the role of chemokine–chemokine receptor interactions in local T‐cell retention. The results show that expression of the CCR10 targeting ligand CCL27 is not only increased during inflammation, but also remains increased several weeks after clinical responsiveness to patch testing. In parallel with increased CCL27 expression, an increased number of infiltrating cells could still be detected in skin that, clinically, had returned to normal 21 days after patch testing. These persisting cells were characterized as CD4+ cells expressing CCR10, while no CD8+ CCR10+ cells could be detected. The presence of these cells is most likely an allergen‐mediated effect, as increased levels of CCL27 and CCR10 could not be detected 21 days after initiating an irritant contact dermatitis reaction. In contrast to CCL27, increased expression of CXCL9, CXCL10, and CXCL11 could only be observed during the clinically inflammatory phase of ACD. In conclusion, local CCL27‐mediated retention of CCR10+ CD4+ T cells in sites previously challenged by ACD could be responsible for phenomena such as local skin memory observed in retest reactions and flare‐up reactions in which the presence of persisting T cells results in an accelerated inflammatory response upon renewed allergen challenge. Copyright

Nickel-responding T cells are CD4+ CLA+ CD45RO+ and express chemokine receptors CXCR3, CCR4 and CCR10

Background  Whereas T lymphocytes are widely accepted as effector cells determining the pathogenesis of allergic contact dermatitis, contradictory results have been found regarding the roles of different T‐cell subsets. The use of various experimental models, involving long‐term cultured T‐cell lines or clones, may explain these contradictory results.

Human growth factor (huGRO), interleukin-8 (IL-8) and interferon-γ-inducible protein (γ-IP-10) gene expression in cultured normal human keratinocytes

HuGRO, IL-8 and γ-IP-10 belong to a recently described superfamily of genes encoding a group of cytokines with inflammatory, growth regulating and/or leukocyte chemotactic properties (chemokines). We studied huGRO, IL-8 and γ-IP-10 gene expression in unstimulated and stimulated (TNFα, INFγ, TNFα + IFNγ, IL-1Β, PMA and LPS) normal human keratinocytes by Northern blot analysis. The mRNA for none of the three chemokines was detectable in unstimulated keratinocytes, but considerably elevated levels of huGRO and IL-8 mRNA, but not of γ-IP-10 mRNA, were found in the presence of cycloheximide, indicating that huGRO and IL-8 mRNA, but not γ-IP-10 mRNA, are constitutively produced. γ-IP-10 mRNA was exclusively induced by IFNγ, with a strong and transient rise between 8 and 18 h, and superinduced by the combination of IFNγ and TNFα, indicating marked synergism. Both huGRO and IL-8 mRNA were induced by TNFα and PMA (a strong and transient rise between 2 and 8 h), but not by IFNγ or LPS. The combination of TNFα and IFNγ did not show a synergistic effect. In addition, IL-1Β transiently upregulated huGRO mRNA but failed to induce IL-8 mRNA. Using specific oligonucleotides for α, Β and γ huGRO, TNFα was found to induce all three forms, α and Β to an equal extent and γ to a lesser extent. Our results indicate that there is stimulus-specific transcription of these early response genes which may have important implications for the modulation of cutaneous inflammatory processes.

Double-enzyme conjugates, producing an intermediate color, for simultaneous and direct detection of three different intracellular immunoglobulin determinants with only two enzymes.

A new double-enzyme conjugate was synthesized by coupling alkaline phosphatase (AP) to horseradish peroxidase (HRP). After AP (blue) and subsequent HRP (red) cytochemistry, this new conjugate produced a stable intermediate-colored (violet) product. By coupling this double-enzyme conjugate to an antigen (trinitrophenyl, TNP) or an antibody (anti-mouse immunoglobulin G2a), anti-TNP or -IgG2a-producing cells could be demonstrated as violet cells in spleen sections. This led to the development of a rapid one-step incubation--two-step cytochemical procedure for simultaneous detection of three different determinants in a single tissue section. To demonstrate this novel triple staining method, we coupled three different antigens to, respectively, AP, HRP, and AP-HRP. When spleen sections of immunized animals were incubated with a mixture of these three antigen-enzyme conjugates, we could distinguish antibody-forming cells against each of these three antigens simultaneously as red (HRP), blue (AP), and violet (AP-HRP) cells. The simultaneous detection of three different classes of intracellular antibodies in a single section also proved to be possible with this method. With this study we provide a new direct method for detection of three different intracellular immunoglobulins after a one-step incubation and a two-step standard cytochemical procedure.

Specthophotmetric quantification of cell-cell adherence by an enzyme-linked immuno-cell adhesion assay

In the present study a spectophotometric method was developed to determine the number of T cells binding to human epidermal keratinocytes (KC). In this cell adhesion assay the adherent T cells were detected with a cell-specific monoclonal antibody conjugated to horseradish peroxidase and the coloured substrate quantified in an ELISA reader at 492 nm. A correlation was demonstrated between the number of T cells and the extinction values measured. The enzyme-linked immuno-cell adhesion assay (ELICAA) was used to quantify KC/T lymphocyte adherence in a series of experiments designed to evaluate its reliability and reproducibility. Compared with the 51Cr-labelled adherence assay, the ELICAA was a safe, rapid and accurate method avoiding the use of radioactive material.

The expression of interleukin-8 receptor in untreated and treated psoriasis

The expression of IL-8 in psoriasis has been clearly shown with the use of immunocytochemical, RT-PCR and in situ hybridization methods. The presence of its ligand, the IL-8 receptor, has been demonstrated by the RT-PCR technique. We report here a study of the expression of both IL-8 type A and B receptors by immunohistochemical techniques, using one polyclonal and four monoclonal antibodies. By this technique, we found that the neutrophilic granulocytes express the IL-8 type A receptor, whereas the IL-8 type B receptor was present on the keratinocytes. The type B receptor on the keratinocytes was localized in the suprabasal layers of the epidermis. Following therapy, the expression of the IL-8 type B receptor on the keratinocytes was reduced. This could suggest that IL-8 in psoriasis is involved in the disturbed differentiation rather than in proliferation, probably via an autocrine loop.