Genomic organization, sequence and transcriptional regulation of the human CXCL 11(1) gene.

CXCL 11, encoded by the cDNA sequences designated beta-R1, H-174, or I-TAC, is a CXC chemokine ligand for CXCR3 and assumed to be involved in inflammatory diseases characterized by the presence of activated T-cells. We here describe the genomic organization (four exons interrupted by three introns of 585, 98 and 230 bp) and sequence including 960 bp from the immediate 5'-upstream region of the human CXCL 11 gene. Within the promoter region, consensus sequences for regulatory elements (ISRE, GAS, NF-kappaB) important for cytokine-induced gene transcription were identified. The effect of (pro)inflammatory cytokines on CXCL 11 mRNA expression in monocytic cell lines (THP-1, U937) and primary cultures of dermal fibroblasts and endothelial cells were examined using Northern blot analysis. For these cell types, IFN-gamma was a potent inducer of CXCL 11 transcription, which was synergistically enhanced by TNF-alpha.

Characterization of paraneoplastic pemphigus autoantigens by immunoblot analysis.

We investigated the antigen molecules for six clinically typical cases of paraneoplastic pemphigus (PNP) using immunofluorescence, immunoprecipitation, and immunoblotting. All the PNP sera showed a clear reactivity with transitional epithelia of rat urinary bladder and immunoprecipitated the 250-kD, 230-kD, 210-kD, 190-kD, and 170-kD proteins in various combinations, confirming the diagnosis of PNP. Immunoblot analysis demonstrated slightly different reactivity from that of immunoprecipitation. With immunoblotting of normal human epidermal extract, bovine desmosome preparation, and extract of cultured squamous cell carcinoma cells, all the PNP sera reacted with a characteristic doublet of the 210-kD and 190-kD proteins. However, immunoblotting detected the 250-kD desmoplakin I and the 230-kD bullous pemphigoid antigen less frequently and did not detect the 170-kD protein. Further immunoblot studies indicated that the 210-kD protein is different from desmoplakin II and that the 190-kD protein is most frequently detected by PNP sera. Two of the six PNP sera specifically reacted with the extracellular domain of recombinant pemphigus vulgaris antigen protein, indicating that pemphigus vulgaris antigen may be involved in PNP. In future studies to unravel the complex mechanisms of the PNP antigens, the immunoblot technique may be a useful tool.

Epidermal interferon-gamma inducible protein-10 (IP-10) and monokine induced by gamma-interferon (Mig) but not IL-8 mRNA expression is associated with epidermotropism in cutaneous T cell lymphomas.

Epidermal infiltration by neoplastic CD4+ T cells is a characteristic histologic feature of early stage mycosis fungoides, the most common type of cutaneous T cell lymphoma (CTCL). The mechanisms involved in epidermotropism are unknown. It has been suggested that the CXC chemokines IL-8 and interferon-gamma inducible protein 10 (IP-10) may play a role, but evidence that these chemokines are produced within the epidermis in epidermotropic CTCL is lacking. In this study skin biopsies from 17 CTCL patients, including 12 mycosis fungoides, four pleomorphic CTCL, and one CD8+ CTCL, were investigated for epidermal IL-8 and IP-10 mRNA expression by RNA in situ hybridization. In addition, the expression of monokine induced by gamma-interferon (Mig) mRNA, a CXC chemokine closely related to IP-10, was studied as well. The expression of IL-8 receptors A and B (CXCR1 and CXCR2, respectively) was investigated by immunohistochemistry. The results were correlated with the number and phenotype of epidermotropic T cells. Epidermal expression of IP-10 and Mig mRNA was detected in 10 of 11 and seven of 11 epidermotropic CTCL, respectively, but not in five nonepidermotropic CTCL biopsies or normal human skin. Epidermal IP-10 and Mig mRNA expression correlated with epidermal infiltration of CD4+ T cells, but not of CD8+ T cells. IL-8 mRNA was demonstrated in the epidermis of only two of 15 CTCL biopsies, and was associated, in both cases, with accumulation of neutrophils. Consistently, immunostaining of the (intraepidermal) T cells with antibodies against CXCR1 and CXCR2 was not observed. In conclusion, the results of this study indicate that IP-10, and to a lesser extent Mig, but not IL-8 is involved in the preferential infiltration of neoplastic CD4+ T cells in CTCL.

The CXCR3 activating chemokines IP-10, Mig, and IP-9 are expressed in allergic but not in irritant patch test reactions.

Differentiation between allergic and irritant contact dermatitis reactions is difficult, as both inflammatory diseases are clinically, histologically, and immunohistologically very similar. Previous studies in mice revealed that the chemokine IP-10 is exclusively expressed in allergic contact dermatitis reactions. In the present study, we investigated whether the mRNA expression of IP-10 and the related CXCR3 activating chemokines, Mig and IP-9 are also differentially expressed in human allergic contact dermatitis and irritant contact dermatitis reactions. Skin biopsies from allergic (13 cases) and sodium lauryl sulfate-induced irritant patch test reactions (13 cases), obtained 1-72 h after patch testing, were studied by means of an in situ hybridization technique. Results of chemokine mRNA expression were correlated with clinical scoring, histology, and immunohistochemical data including the proportion of inflammatory cells expressing CXCR3, the receptor for IP-10, Mig, and IP-9, and ICAM-1 and HLA-DR expression on keratinocytes. IP-10, Mig, and IP-9 mRNA were detected in seven of nine allergic contact dermatitis reactions after 24-72 h, but not in sodium lauryl sulfate-induced irritant contact dermatitis reactions. ICAM-1 expression by keratinocytes was only found in allergic contact dermatitis reactions and correlated with chemokine expression. Moreover, up to 50% of the infiltrating cells in allergic contact dermatitis expressed CXCR3, in contrast to only 20% in irritant contact dermatitis reactions. In conclusion, we have demonstrated differences in chemokine expression between allergic contact dermatitis and irritant contact dermatitis reactions, which might reflect different regulatory mechanisms operating in these diseases and may be an important clue for differentiation between allergic contact dermatitis and irritant contact dermatitis reactions.

Human IP-9: A keratinocyte-derived high affinity CXC-chemokine ligand for the IP-10/Mig receptor (CXCR3).

Chemokines and their receptors play a crucial part in the recruitment of leukocytes into inflammatory sites. The CXC chemokines IP-10 and Mig are selective attractants for activated (memory) T cells, the predominant cell type in skin infiltrates in many inflammatory dermatoses. The selectivity for activated T cells can be explained by the fact that both chemokines exert their effects through a common receptor, CXCR3, which is nearly exclusively expressed on activated T cells. The aim of this study was to identify biologically active CXCR3 ligands produced by keratinocytes. To that end, Chinese hamster ovary cells expressing a cDNA encoding CXCR3 were challenged with proteins obtained from interferon-gamma stimulated keratinocytes and subsequently monitored for effects on second messenger systems. By this approach we were able to isolate IP-10 and Mig, and in addition identified a novel highly potent ligand for the CXCR3 receptor, designated interferon-gamma-inducible protein-9, which proved to be chemotactic for activated T cells expressing CXCR3. Protein sequence and mass spectrometric analysis followed by molecular cloning of the cDNA encoding interferon-gamma-inducible protein-9, revealed that interferon-gamma-inducible protein-9 is a CXC chemokine with a molecular mass of 8303 Da. From a GenBank database query it became clear that interferon-gamma-inducible protein-9 is in fact the protein encoded by the cDNA sequence also known as beta-R1, H174 or I-TAC. In situ hybridization experiments showed that interferon-gamma-inducible protein-9 mRNA is expressed by basal layer keratinocytes in a variety of skin disorders, including allergic contact dermatitis, lichen planus, and mycosis fungoides suggesting a functional role for this chemokine in skin immune responses.

Periodate or glutaraldehyde for preparing peroxidase conjugates?

Conjugates of horseradish peroxidase with antibodies (anti-human IgG (H + L)) or their Fab' fragments were prepared according to the newest modification of the periodate (P-) method or the two-step glutaraldehyde (G-) method. The conjugates were analysed by gel chromatography and subsequently tested in three different applications. For tissue immunohistochemistry on sections of lupus erythematosus skin, G-conjugates were preferred to polymeric P-conjugates. In ELISA for detection of human antibodies against penicillin P-conjugates were superior to G-conjugates. For the detection of surface Ig on lymphoid cells both types of conjugate were more or less equally suitable. A scheme for the most suitable combinations of method of preparation and field of application is given.

Spectophotometric 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.

A rapid and simple hapten conjugation method for monoclonal antibodies to be used in immunoenzyme single and double staining procedures.

A rapid and simple method was developed for the haptenization of monoclonal antibodies (MAbs), to be used in immunoenzyme single and double staining techniques. Using this method minute amounts of MAbs can be haptenized without purification of the antibody or removal of the excess hapten. The haptenized MAb can be ready for use with 2.5 h. The method consists of a direct incubation of the antibody with the haptenizing agent and subsequent addition of an amino acid solution to stop the reaction. Commonly available reagents were tested, of which dinitrofluorobenzene, trinitrobenzene sulfonic acid and an oxazolone derivative gave the best results. The procedure was evaluated by using MAbs directed against lymphoid cell surface membrane antigens in an indirect immunoenzyme staining on frozen sections using peroxidase or alkaline phosphatase-conjugated anti-hapten antibodies as second step antibody. It was found that those MAbs, which show good staining results in conventional indirect immunoenzyme procedures, can also be used successfully after haptenization in single as well as double staining procedures. Combination of haptenized and biotynilated MAbs gave good results when weakly reactive MAbs had to be included in immunoenzyme double staining.

Peroxidase-conjugate chromatography isolation of conjugates prepared with glutaraldehyde or periodate using polyacrylamide-agarose gel.

Fractionation abilities of polyacrylamide-agarose gel (Ultrogel) and dextran gel (Sephadex) column chromatography were compared in isolating horseradish peroxidase conjugates, prepared using two different methods. Utrogel AcA-44 provides an efficient separation of monomer conjugated and nonconjugated immunoglobulins resulting from the two-step glutaraldehyde procedure, Sephadex G-200 does not. Both types of columns eluted the polymer conjugates resulting from the periodate procedure in the void volume; these were hardly isolated from the small amount of monomer conjugate. Unreacted horseradish peroxidase, present in very low quantities after the efficient periodate method and in large amounts after the glutaraldehyde procedure, was separated by both gel types.

A comparative study of horseradish peroxidase conjugates prepared with a one-step and a two-step method.

In this study we compared horseradish peroxidase (HRP)-labeled rabbit antihuman immunoglobulin G (IgG) conjugates, prepared by a one-step and a two-step method. Glutaraldehyde was used as a cross-linking agent. Two methods were used for removing unconjugated HRP: Sephadex G-200 gel chromatography and ammonium sulfate precipitation. The conjugates were characterized immunologically, immunochemically and enzymatically. The immunohistoenzymic properties of the conjugates were tested on unfixed cryostat sections of the skin of patients with chronic discoid lupus erythematosus. The influence of the presence of unconjugated HRP and unconjugated IgG was studied. Optimal results were obtained with conjugates prepared by a two-step method. Removing unconjugated HRPimproved the immunohistoenzymic properties of the conjugates. Conjugated and unconjugated IgG could be separated by Sephadex G-200 gel chromatography.

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.

Direct immunocytochemistry with a horseradish peroxidase-conjugated monoclonal antibody against substance P.

The procedure for the isolation and conjugation of the anti-substance P monoclonal antibody NC1/34 with the enzyme horseradish peroxidase (HRP) is described. This resulted in a molecular complex of monoclonal antibody/HRP of 1:1. This conjugate was of approximately 400,000 daltons, as estimated by gel chromatography. Practically all the isolated antibody was coupled to HRP. The conjugate was tested both in a model system where CNBr-activated Sepharose beads were coupled to substance P and on fixed tissue preparations from the rat spinal cord and medulla oblongata. The conjugate revealed staining in nerve fibers in areas known to contain substance P. The best immunohistochemical results were obtained by prolonged incubations at 12 degrees C in the presence of 0.1% Triton X-100. The preabsorption of the conjugate with substance P obliterated the reaction.

The effect of occlusion of the skin with transdermal therapeutic system on Langerhans' cells and the induction of skin irritation.

Sensitization to drugs in transdermal therapeutic systems is a common unwanted event. We investigated whether prolonged occlusion of the skin causes skin irritation, and in this way might play a role in the possible induction of sensitization to drugs from these systems. Occlusion was effected with a placebo transdermal therapeutic system and silver-patch test in a time span ranging from eight hours to seven days in five groups of five volunteers. Skin irritation was judged on clinical aspects, histopathologic and immunofluorescence findings, and changes in the Langerhans' cell systems. The results indicate that occlusion with the systems used in this experiment provokes only slight or no skin irritation.

Bullous pemphigoid and epidermolysis bullosa acquisita. Differentiation by fluorescence overlay antigen mapping.

BACKGROUND AND DESIGN: From previous studies, we concluded that the fluorescence overlay antigen mapping (FOAM) technique could be of value to the differential diagnosis of the acquired subepidermal bullous skin disorders, bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA). In these diseases, ultrastructural identification of the site of skin-bound IgG deposits at the epidermal basement membrane zone (EBMZ) may be essential to the correct diagnosis. Since ultrastructural studies are more expensive, time-consuming, and less widely available than immunofluorescence, we addressed the question of whether the FOAM technique can reliably identify the site of IgG deposits at the EBMZ, and distinguish BP from EBA. For this purpose, the technique was applied to perilesional skin from seven patients with BP and six with EBA, using computer-aided imaging of red-stained type VII collagen and green-stained IgG, according to previous findings. RESULTS: Digitized multicolor FOAM images of perilesional skin from patients with BP showed nonoverlap band patterns of green-stained lamina lucida IgG deposits (ultrastructurally proven) and red-stained type VII collagen. By contrast, FOAM images of EBA skin typically showed overlap patterns of green-stained sublamina densa IgG deposits and red-stained type VII collagen. These findings were observed also in skin tissue stored in Michel's transport medium or stored frozen for 15 years. CONCLUSIONS: The computer-aided FOAM technique may have great potential in distinguishing between IgG deposits above (BP) and just below (EBA) the lamina densa of the EBMZ in skin tissue. The technique is not as simple as saline-split skin methodology but offers more flexibility, and it certainly is quicker and less expensive than electron microscopy. Furthermore, the use of digitized fluorescence images offers improved possibilities for evaluating the various "linear" patterns of immune reactant deposition at the EBMZ in subepidermal bullous autoimmune skin diseases.

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

HuGRO, IL-8 and gamma-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 gamma-IP-10 gene expression in unstimulated and stimulated (TNF alpha, INF gamma, TNF alpha + IFN gamma, IL-1 beta, 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 gamma-IP-10 mRNA, were found in the presence of cycloheximide, indicating that huGRO and IL-8 mRNA, but not gamma-IP-10 mRNA, are constitutively produced. gamma-IP-10 mRNA was exclusively induced by IFN gamma, with a strong and transient rise between 8 and 18 h, and superinduced by the combination of IFN gamma and TNF alpha, indicating marked synergism. Both huGRO and IL-8 mRNA were induced by TNF alpha and PMA (a strong and transient rise between 2 and 8 h), but not by IFN gamma or LPS. The combination of TNF alpha and IFN gamma did not show a synergistic effect. In addition, IL-1 beta transiently upregulated huGRO mRNA but failed to induce IL-8 mRNA. Using specific oligonucleotides for alpha, beta and gamma huGRO, TNF alpha was found to induce all three forms, alpha and beta to an equal extent and gamma to a lesser extent.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Allergic reactions, "spillover' reactions, and T-cell subsets.

A strong positive, allergic patch-test reaction was elicited in 15 patients with an established allergy for a particular allergen. Patches with a marginally irritating concentration of sodium lauryl sulfate (SLS) were applied at fixed distances. The SLS patch situated adjacent to the allergic reaction was significantly enhanced in 12 of 15 patients (P less than 0.01) compared to more distant SLS reactions ("spillover'). Only quantitative differences were observed in the histologic pictures of the different types of reaction. The infiltrate consisted of lymphocytes and histiocytes, mainly located perivascular in the upper dermis. T-cell subsets were assessed with monoclonal antibodies using an immunoperoxidase technique. The distribution of the different T cells was the same for both reaction types. T cells located outside the perivascular infiltrates (e.g., in the epidermal vesicles) were OKT-8-positive (cytotoxic/suppressor T lymphocytes). Immunofluorescence examination did not show different patterns for the allergic or "enhanced toxic' reactions with regard to the presence of immunoglobulins and complement. The "spillover' phenomenon may cause false-positive patch-test reactions.

Chemokine IP-10 expression in cultured human keratinocytes.

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-gamma, 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-gamma and TNF-alpha 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-gamma, TNF-alpha, or a combination of IFN-gamma and TNF-alpha. 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.

Effect of calcitriol on growth, differentiation, chemokine mRNA expression of cultured keratinocytes and on keratinocyte-T cell binding.

Calcitriol has recently been shown to be effective against psoriasis. However, its mode of action is not exactly known. The present study focused on the influence of calcitriol on growth, differentiation, chemokine mRNA and ICAM-1 mRNA expression of keratinocytes (KC) and on the binding of T-cells to keratinocytes. In vitro studies showed that calcitriol has a strong anti-proliferative effect and induces terminal differentiation. gamma-IP-10 and ICAM-1 mRNA were induced by gamma-IFN, an induction not influenced by calcitriol. Moreover, the functional expression of ICAM-1 on the KC cell surface as measured by a cell adhesion assay, was not influenced either. IL-8 and huGRO mRNAs were constitutively produced in KC, as was demonstrated after incubation with cycloheximide. Up-regulation of both IL-8 and huGRO mRNA by IL-1 alpha was also not affected by calcitriol. It is concluded that calcitriol has a strong antiproliferative activity and does not interfere with KC responsiveness to gamma-IFN and IL-alpha induced chemokine expression or with the adhesion of T-cells to keratinocytes.

Detection of specific anti-hapten antibody-producing hybridoma cells in tissue sections of spleen and liver by hapten-enzyme conjugates using an immunoenzyme approach.

A method is presented for the detection of hybridoma cell growth in vivo by making use of the specific antigen (hapten) recognition properties of the produced monoclonal antibodies. Horseradish peroxidase (HRP)-labeled antigen was used for demonstration of intracellular specific antibodies in hybridoma cells. It appeared that intravenously injected anti-penicilloyl-producing hybridoma cells developed mainly in the red pulp of the spleen; to a lesser extent they were found in the liver.