Increased expression of the interleukin-36 cytokines in lesions of hidradenitis suppurativa.

BACKGROUND: Hidradenitis suppurativa (HS) is a recalcitrant chronic skin disease with poorly understood immunopathogenic mechanisms. Previous studies reported that the interleukin-36 (IL-36) cytokines [IL-36α, IL-36ß, IL-36γ and IL-36 receptor antagonists (IL-36RA)] are important players in the pathogenesis of psoriasis (PS). OBJECTIVE: We aim to determine whether the IL-36 cytokines are upregulated in patients with HS. For this purpose, we analysed local expression and systemic levels of the IL-36 cytokines in patients with HS and compared the results to healthy donors and patients with PS. METHODS: Skin biopsies from healthy donors and HS and PS patients were analysed for expression of the IL-36 cytokines by immunohistochemistry and semiquantitative real-time PCR. The enzyme-linked immunosorbent assay (ELISA) was used to measure systemic levels of the IL-36 cytokines in the serum of the three donor groups. RESULTS: The agonists IL-36α, IL-36ß and IL-36γ were found to be upregulated in HS both systemically and lesionally, while the IL-36RA was not differently regulated in comparison to healthy donors. CONCLUSION: Our findings suggest that the agonistic IL-36 isoforms are upregulated in HS. The relevance of the enhanced production of IL-36 cytokines in HS pathogenesis remains to be determined.

Innate immune cells express IL-17A/F in acute generalized exanthematous pustulosis and generalized pustular psoriasis.

Acute generalized exanthematous pustulosis (AGEP) and generalized pustular psoriasis (GPP) are rare pustular skin disorders with systemic involvement. IL-17A/F is a proinflammatory cytokine involved in various neutrophilic inflammatory disorders. Here we show that IL-17A/F is highly expressed by innate immune cells such as neutrophils and mast cells in both AGEP and GPP.

Heat-killed bacillus Calmette-Guérin and Mycobacterium kansasii antigen 85B combined vaccination ameliorates dermatitis in a mouse model of atopic dermatitis by inducing regulatory T cells.

BACKGROUND: Atopic dermatitis (AD) is a recurrent inflammatory skin disease characterized by dominant T-helper (Th) 2 cytokine response. Bacillus Calmette-Guérin (BCG) has been used for preventing tuberculosis, and is regarded as a strong Th1 cytokine inducer. Antigen (Ag) 85B is a secretory protein present in Mycobacterium species that induces Th1 cytokine production. OBJECTIVES: We investigated the effects of combined vaccination of heat-killed BCG (hkBCG) and Mycobacterium kansasii Ag85B in an AD mouse model. METHODS: For the AD model, keratin 14 promoter-derived caspase-1 overexpressing mice (KCASP1Tg) were used. The mice received a combination therapy of hkBCG at age 3 weeks and Ag85B twice weekly for 11 weeks from the 4th week; Ag85B monotherapy from the 4th week; hkBCG monotherapy at the 3rd week; or control saline. Areas of skin lesions, cytokine mRNA expression and serum interleukin (IL)-18 and immunoglobulin (Ig) E levels were analysed. Inducible Foxp3+ regulatory T cells (iTreg), IL-10-producing T cells (Tr1), and interferon (IFN)-γ/IL-4/IL-17-producing T cells were evaluated in the spleen. RESULTS: Saline-treated mice and hkBCG monotherapy mice spontaneously developed severe dermatitis. However, combined therapy with hkBCG and Ag85B significantly suppressed the development of skin lesions and mast cell infiltrations. Elevations of the serum IgE and IL-18 levels were significantly suppressed with combined therapy. Mice treated with hkBCG and Ag85B had a normal number of iTreg in the spleen, and decreased number of both IL-4- and IL-17-producing CD4+ T cells. The effect of Ag85B monotherapy was limited. CONCLUSIONS: Combined vaccination with hkBCG and Ag85B decreases AD skin lesions by inducing regulatory T cells, suggesting that this vaccination is a potent and novel therapeutic strategy for AD.

1,24-Dihydroxyvitamin D3 (tacalcitol) prevents skin T-cell infiltration.

BACKGROUND: 1,24-Dihydroxyvitamin D3 (tacalcitol), a vitamin D(3) compound, has been used to treat T cell-mediated inflammatory skin diseases such as psoriasis, prurigo and vitiligo. The best-known mechanism of action of this compound is inhibition of the abnormal proliferation of keratinocytes and subsequent maturation; however, its effects on skin T-cell recruitment have not yet been evaluated. Cutaneous lymphocyte-associated antigen (CLA), a surface glycoprotein expressed on T cells, plays a critical role in skin T-cell infiltration. We recently reported that 1,25-dihydroxyvitamin D3 inhibits skin infiltration of CD4+ T cells by suppressing CLA expression on T cells. OBJECTIVES: In this study, we investigated the effect of tacalcitol on CLA epitope decoration and on the levels of gut or lymph node homing receptor expression in human T cells. METHODS: We cultured human T cells with tacalcitol and analysed the effect on CLA expression and skin-homing ability, and evaluated glycosyltransferase mRNAs. We also performed an in vivo study using an antigen-dependent delayed-type hypersensitivity (DTH) mouse model and investigated the effect of tacalcitol on skin-infiltrating CD4+ T cells. RESULTS: Tacalcitol downregulated the expression of CLA and, in parallel, the E- and P-selectin ligand function; however, it exerted no effect on other homing receptors. Subcutaneously and intraperitoneally administered tacalcitol downregulated skin infiltration of effector CD4+ T cells in an in vivo DTH mouse model. CONCLUSIONS: These findings suggest that tacalcitol reduces skin inflammation by partially downregulating CLA expression levels.

IL-4/IL-13 antagonist DNA vaccination successfully suppresses Th2 type chronic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a chronic disease with a Th2-type-cytokine dominant profile. Several cytokines and related peptides have been used for the treatment of AD but they were ineffective because of their limited biological half-life. We have recently developed a highly efficient mouse dominant negative interleukin (IL)-4/IL-13 antagonist (IL-4DM), which blocks both IL-4 and IL-13 signal transductions. OBJECTIVE: To examine the effects of IL-4DM in vivo in an AD model induced by the repeated exhibition of oxazolone (OX). METHODS: Plasmid DNA was injected intraperitoneally to cause an experimental AD-like dermatitis. The effect was evaluated by ear thickness, histological findings, and mast cells counts in the inflamed skin. The plasma IgE and histamine levels were measured. Cytokine production in skin and splenocytes were also analysed. RESULTS: Mice treated with control plasmid developed marked dermatitis with mast cells and eosinophil infiltration, and had increased plasma IgE and histamine levels with a Th2 type splenocyte cytokine profile. Treatment with mouse IL-4 DNA augmented the ear swelling and thickness with an increased dermal eosinophil count, plasma histamine level, and production of splenocyte IL-4. However, IL-4DM treatment successfully controlled the dermatitis, decreased the mast cell and eosinophil count, and suppressed plasma IgE and histamine levels. Splenocytes produced an increased level of IFN-gamma. CONCLUSION: These data showed that the simultaneous suppression of IL-4/IL-13 signals successfully controlled Th2-type chronic dermatitis. IL-4DM DNA treatment is a potent therapy for AD and related diseases.

Cytokeratin expression in subungual squamous cell carcinoma.

Cytokeratin expression in subungual squamous cell carcinoma was investigated in order to evaluate the origin and state of differentiation of the tumour. The tumour nests contained cytokeratin 14, 16 and 17, which were also expressed in the nail bed. Therefore, cytokeratin expression in subungual squamous cell carcinoma may reflect its indolent clinical prognosis.

Human artificial chromosome (HAC) vector provides long-term therapeutic transgene expression in normal human primary fibroblasts.

Human artificial chromosomes (HACs) segregating freely from host chromosomes are potentially useful to ensure both safety and duration of gene expression in therapeutic gene delivery. However, low transfer efficiency of intact HACs to the cells has hampered the studies using normal human primary cells, the major targets for ex vivo gene therapy. To elucidate the potential of HACs to be vectors for gene therapy, we studied the introduction of the HAC vector, which is reduced in size and devoid of most expressed genes, into normal primary human fibroblasts (hPFs) with microcell-mediated chromosome transfer (MMCT). We demonstrated the generation of cytogenetically normal hPFs harboring the structurally defined and extra HAC vector. This introduced HAC vector was retained stably in hPFs without translocation of the HAC on host chromosomes. We also achieved the long-term production of human erythropoietin for at least 12 weeks in them. These results revealed the ability of HACs as novel options to circumvent issues of conventional vectors for gene therapy.

Molecular cloning of a novel receptor-type protein tyrosine phosphatase from murine fetal liver.

A cDNA fragment encoding a novel tyrosine phosphatase (PTPase), termed ptpf, was isolated from day 11.5 mouse fetal liver using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers. The 5.5-kb cDNA encoding the complete coding region was isolated from an adult mouse kidney cDNA library. This cDNA contained a single open reading frame (ORF) encoding a predicted 1436-amino-acid protein with a molecular mass of 161,150 Da. Sequence analysis revealed that PTPf was homologous to PTPmu and PTPkappa, and a putative receptor-type PTPase. Northern blotting analysis of adult mouse mRNA indicated the existence of four major ptpf transcripts of approximately 10, 6, 3 and 2.7 kb, and these transcripts were expressed in a tissue-specific manner. During embryogenesis, only the 6-kb transcript was detected.

In vitro long-term culture of human primitive hematopoietic cells supported by murine stromal cell line MS-5.

When Lin-CD34+CD38- cells from normal human cord blood were cocultured with MS-5, colony forming cells were maintained for over 8 weeks. Prevention of contact between MS-5 and Lin-CD34+CD38- cells by using a membrane filter was negligible for this activity, indicating that the activity of MS-5 on human primitive hematopoietic cells may be due to soluble factor(s) secreted from MS-5. We tried to purify this activity by a [3H]TdR incorporation assay. The activity was found in 150 kD fraction and was neutralized with anti-mSCF (stem cell factor) antibody. Another 20-30 kD fraction synergized with mSCF to stimulate the growth of Lin-CD34+CD38- cells but failed alone. This fraction supported the growth of the G-CSF (granulocyte-colony stimulating factor)-dependent cell line FD/GR3, FDC-P2 transfected with mG-CSF receptor cDNA. This synergy was canceled in the presence of soluble mG-CSF receptor. Addition of anti-mSCF antibody and soluble mG-CSF receptor to the culture completely abrogated the activity of MS-5-culture supernatant. These results indicate the activity of MS-5 on Lin-CD34+CD38- cells is due to synergistic effect of mSCF and mG-CSF.

Granulocyte-colony stimulating factor and stem cell factor are the crucial factors in long-term culture of human primitive hematopoietic cells supported by a murine stromal cell line.

The findings that murine marrow stromal cell line MS-5 supported the proliferation of human lineage-negative (Lin-) CD34+CD38- bone marrow cells in long-term culture have been reported. In this study, we analyzed this proliferating activity of MS-5-conditioned medium (CM) on human primitive hematopoietic cells. When Lin-CD34+CD38- cells of normal human cord blood cells were co-cultured with MS-5, colony forming cells (CFCs) were maintained over 7 weeks in vitro. Prevention of contact between MS-5 and Lin-CD34+CD38- cells by using membrane filter (0.45 micron) was negligible for this activity. This indicated that the activity of MS-5 on human primitive hematopoietic cells is a soluble factor(s) secreted from MS-5, which is not induced by the contact between MS-5 and Lin-CD34+CD38- cells. We tried to purify this soluble activity. An active material with a molecular weight of about 150 kDa, determined by gel filtration chromatography, solely supported the growth of Lin-CD34+CD38- cells and Mo7e, a human megakaryocytic cell line. This activity not only reacted with anti-mouse stem cell factor (mSCF) antibody on Western blots, but it was also neutralized in the presence of anti-mSCF antibody. Another active material with a molecular weight of about 20-30 kDa synergized with mSCF to stimulate the growth of Lin-CD34+CD38- cells but failed to do so alone, although this synergy was inhibited in the presence of soluble mouse granulocyte-colony stimulating factor (mG-CSF) receptor, which is a chimeric protein consisting of the extracellular domain of mG-CSF receptor and the Fe region of human IgG1. In addition, the latter molecule supported the growth of the G-CSF dependent cell line FD/GR3, which is a murine myeloid leukemia cell line, FDC-P2, transfected with mG-CSF receptor cDNA. Adding of anti-mSCF antibody and soluble mG-CSF receptor to the culture completely abrogated the activity of MS-5-CM. Recombinant (r) mSCF and rmG-CSF had synergistic activity on the growth of Lin-CD34+CD38- cells. These results indicated that the activity on Lin-CD34+CD38- cells included in MS-5-CM is based upon the synergistic effects of mSCF and mG-CSF.

An Escherichia coli curved DNA-binding protein whose expression is affected by the stationary phase-specific sigma factor sigma S.

From Escherichia coli, a DNA-binding protein that preferentially recognizes a curved DNA sequence was isolated and shown to correspond to one that has recently been reported as a binding protein for the replication origin of the E. coli chromosome, named Rob. Here, a rob promoter-lacZ transcriptional fusion was constructed on the chromosome, and used to demonstrate that the expression of rob is notably enhanced at the onset of stationary phase in Luria-broth and also under certain growth conditions in a minimal medium, such as glucose- and phosphate-starvation medium. It was further shown that this growth condition-dependent expression of rob is notably reduced in a null mutant for the stationary phase-specific sigma subunit of RNA polymerase, sigma s, although sigma s-independent expression of rob was significant during the logarithmic growth phase. Furthermore the rob null mutant was found to exhibit, as compared with the wild-type, an altered profile of protein synthesis, particularly at the very late stationary phase.

A study of the double mutation of dnaJ and cbpA, whose gene products function as molecular chaperones in Escherichia coli.

The CbpA protein is an analog of the DnaJ molecular chaperone of Escherichia coli. To gain insight into the function of CbpA, we examined the nature of a cbpA null mutation with special reference to those of dnaK and dnaJ null mutations. In particular, the cbpA dnaJ double-null mutant was found to exhibit severe defects in cell growth, namely, a very narrow temperature range for growth, a defect in cell division, and susceptibility to killing by carbon starvation. These phenotypes are very similar to those reported for dnaK null mutants but not to those of dnaJ null mutants. Our results are best interpreted by assuming that CbpA is capable of compensating for DnaJ for cell growth and thus that the function(s) of CbpA is closely related to that of DnaJ.

An analogue of the DnaJ molecular chaperone whose expression is controlled by sigma s during the stationary phase and phosphate starvation in Escherichia coli.

The Escherichia coli CbpA protein appears to be an analogue of the molecular chaperone, DnaJ, as judged from not only its structure but also its possible function. The expression of cbpA, however, was not significantly affected by up-shift of the growth temperature. Remarkably, it was found that the expression of cbpA was induced under certain growth conditions, such as the entry of cells into stationary phase, or growth in a phosphate-limited medium. Such conditional expression of cbpA was regulated at the transcriptional level in a sigma s-dependent manner. The structure of this sigma s-dependent cbpA promoter was clarified by determining its transcription start site. The cbpA promoter region was found to contain an unusual DNA structure (i.e. DNA curvature). From these results, it was suggested that, in contrast to DnaJ, CbpA may function as a molecular chaperone in an adaptive response to environmental stresses other than heat shock.

An analogue of the DnaJ molecular chaperone in Escherichia coli.

Escherichia coli DnaJ functions as a typical molecular chaperone in coordination with other heat shock proteins such as DnaK and GrpE in a variety of cellular processes. In this study, it was found that E. coli possesses an analogue of DnaJ, as judged from not only its primary structure but also its possible function. This protein, named CbpA (for curved DNA-binding protein), was first identified as a DNA-binding protein that preferentially recognizes a curved DNA sequence. Cloning and nucleotide sequencing of the gene encoding CbpA revealed that the predicted product is very similar to DnaJ in amino acid sequence: overall identity is 39%. The cbpA gene functions as a multicopy suppressor for dnaJ mutations. The mutational lesions characteristic of a dnaJ null mutant--namely, temperature sensitivity for growth and defects in lambda phage and mini-F DNA replication--were all restored upon introduction of the cbpA gene on a multicopy plasmid. An insertional mutant of cbpA was also isolated, which showed no noticeable phenotype, particularly with regard to temperature sensitivity for growth. However, when this cbpA::kan allele was combined with the dnaJ null allele, the resultant strain was unable to grow at 37 degrees C, at which strains carrying each mutation alone could grow normally. These genetic results are interpreted as meaning that the function(s) of CbpA in E. coli is closely related to that of DnaJ.

Autoregulatory expression of the Escherichia coli hns gene encoding a nucleoid protein: H-NS functions as a repressor of its own transcription.

The Escherichia coli nucleoid protein, H-NS (or H1a), appears to influence the regulation of a variety of unrelated E. coli genes and operons. To gain an insight into the regulation of the hns gene itself, we constructed in this study a hns-lacZ transcriptional fusion gene and inserted a single copy at the att lambda locus on the E. coli chromosome. Expression of hns transcription appeared to be moderately regulated in a growth phase-dependent manner. It also emerged that hns transcription is under negative autoregulation, at least in the logarithmic growth phase. The results of in vitro transcription experiments confirmed that H-NS functions as a repressor for its own transcription. Thus, H-NS was shown to exhibit relatively high affinity for the DNA sequence encompassing the hns promoter region, as compared with a non-specific sequence. These results support the view that the nucleoid protein, H-NS, can function as a transcriptional regulator.