Cyclic nucleotide phosphodiesterase 4 subtypes are differentially expressed by primary keratinocytes and human epidermoid cell lines.

Cyclic AMP phosphodiesterase (PDE) type 4 has been the subject of considerable interest as a potential molecular target for treating cutaneous inflammatory and allergic disorders; however, little is known regarding the expression of PDE 4 isogenes in human keratinocytes, the predominant cell type present in the epidermis. In this study, we investigated the expression of PDE 4 subtypes in epidermal cells (primary keratinocytes derived from breast skin, epidermoid cell lines A431, KB, and HaCaT) using reverse transcriptase polymerase chain reaction. Analysis of the amplified cDNA showed that there were differences in the expression of PDE 4 isogenes in the epidermal cells. Constitutive expression of the PDE 4 isozymes was detected in untreated epidermal cells at different levels. Transcripts for PDE 4B and 4D were abundantly expressed in breast skin-derived keratinocytes, whereas transcripts for PDE 4A, 4C, and 4D were present in HaCaT cells and transcripts for all the PDE 4 isotypes were present in A431 and KB. PDE 4A and 4C were detectable in HaCaT cells in equal amounts, but PDE 4C was only marginally expressed in the other cells. Of the four isogenes, PDE 4D was abundantly expressed in all the cells. Elevation of intracellular levels of cAMP by forskolin increased the expression of all the hPDE 4 isogenes 2-3-fold as revealed by semiquantitative reverse transcriptase polymerase chain reaction. Western blot analysis of extracts from control and forskolin or dibutyryl cAMP-treated A431 and KB cells demonstrated the presence of proteins with a molecular mass identical to that corresponding to recombinant human PDE 4B. Together, these findings show that PDE 4 isogenes are expressed in keratinocytes to a different degree and that their expression can be modulated by intracellular levels of cAMP.

Glucosylceramide synthase activity in murine epidermis: quantitation, localization, regulation, and requirement for barrier homeostasis.

Ceramides, which derive from the hydrolysis of glucosylceramide (GlcCer), are the predominant lipid species in the stratum corneum and are critical for epidermal permeability barrier homeostasis. UDP-glucose:ceramide glucosyltransferase (GlcCer synthase) (EC 2.4.1.80) catalyzes the glucosylation of ceramide to form GlcCer. Recently, we demonstrated a progressive increase in GlcCer synthase expression during fetal barrier development, while others have reported increased GlcCer synthase activity with differentiation of cultured human keratinocytes. To further delineate the role of GlcCer synthase in barrier homeostasis, we determined GlcCer synthase activity and localization in hairless mouse epidermis, both under basal conditions and after acute barrier perturbation. Under basal conditions, GlcCer synthase activity localizes predominantly (approximately 80%) to the dithiothreitol-separated outer epidermis; i.e., 6.2+/-0.6 versus 1.2+/-0.1 pmol/min/mg for outer vs. lower epidermis, respectively (P < 0.0001). Although acute barrier disruption does not up-regulate epidermal GlcCer synthase activity at any time point up to 24 h, GlcCer synthase is required for barrier homeostasis: topical d,1-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (P4), a specific GlcCer synthase inhibitor, applied immediately after acute barrier disruption, causes a delay in barrier recovery attributable to specific enzyme inhibition. These findings demonstrate first, that GlcCer synthase activity predominates in the outer epidermis, consistent with an increased formation of GlcCer during barrier ontogenesis and maintenance. Second, GlcCer synthase activity is required for normal permeability barrier homeostasis. Third, baseline epidermal GlcCer synthase activity appears to accommodate acute challenges to the barrier.

Selective inhibition of interleukin-1 beta gene expression in activated RAW 264.7 macrophages by interferon-gamma.

The ability of interleukin-1 (IL-1) to activate epidermal cell populations supports its role as a key cytokine in the pathogenesis of a number of inflammatory skin diseases. In the present study, we have examined the effect of interferon (IFN)-gamma on the expression of the IL-1 beta gene in mouse RAW 264.7 macrophages activated by lipopolysaccharide (LPS) plus tumor necrosis factor (TNF)-alpha. Incubation of macrophages with both LPS and TNF-alpha resulted in the expression of both IL-1 beta and inducible nitric oxide synthase (iNOS) mRNA transcripts and increased the release of IL-1 beta protein and nitrite production in culture supernatants. Addition of IFN-gamma up-regulated the expression of the iNOS gene in cells activated by LPS + TNF-alpha, but significantly suppressed the induction of IL-1 beta gene expression in a dose-dependent manner. The suppression required neither de novo protein synthesis nor involved destabilization of the mRNA transcripts. Together, these findings suggest that IFN-gamma can be an important regulatory cytokine in a chronic inflammatory site and may explain its purported anti-inflammatory effects in certain dermatological diseases.

Genomic structure of NKG5, a human NK and T cell-specific activation gene.

We reported previously the isolation of a cDNA clone, designated NKG5, encoding a secreted protein that is expressed only in natural killer and T cells and is strongly upregulated upon cell activation. In this report we have isolated the NKG5 gene from a human placental genomic library and sequenced the gene and two kilobases of 5'-flanking DNA. Comparison with the cDNA sequence reveals that the NKG5 gene consists of five exons and four introns. Intron 1 contains a DNA segment that was reported to occur as an exon in 519, a closely related cDNA clone that was isolated from a T-cell library. This result indicates that NKG5 and 519 are alternative splicing products of a single gene. The 5'-flanking region of the NKG5 gene was analyzed for homology with the promoter regions of cytokines and other activation-induced genes showing lymphocyte-specific expression. Several segments displaying sequence similarity were identified. We also identified numerous sequence elements that have strong similarity to known binding sites for transcriptional regulatory proteins including T cell-specific and activation-specific regulatory factors. These findings are consistent with the cell-specific expression and the tight regulatory control that is observed for the NKG5 gene.

Specific inhibition of mRNA accumulation for lymphokines in human T cell line Jurkat by mycobacterial lipoarabinomannan antigen.

The immunomodulatory effect of Mycobacterium tuberculosis-derived lipoarabinomannan (LAM) on mitogen/antigen-induced expression of mRNAs for a number of cytokines in human monocytic cell line Mono-Mac-6 and in T cell line Jurkat was investigated. Interestingly, LAM exhibited a down-regulatory effect on the accumulation of mRNAs for IL-2, IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-2 receptor alpha (IL-2R alpha) in T cells co-stimulated with phytohaemagglutinin-P (PHA) and 4 beta-phorbol-12-myristyl-13-acetate (PMA). In human Mono-Mac-6 cells. LAM has a weak inhibitory effect on the lipopolysaccharide (LPS)-induced mRNA accumulation for IL-1 beta, a slight stimulatory effect on mRNAs accumulation for IL-8 and tumour necrosis factor-alpha (TNF-alpha), but clearly no effect on mRNA accumulation for intercellular adhesion molecule-1 (ICAM-1). These findings imply that LAM may contribute to the immunologic defects associated with a number of mycobacterial infections by modulating these mediators.

Serum antibodies against peripheral nervous system antigens in leprosy.

Since antibodies against peripheral nervous system (PNS) antigens may play a pathogenetic role in the mechanism of nerve damage in leprosy, sera from leprosy patients and contacts were investigated for anti-PNS antibodies by ELISA and immunoblot. In ELISA, elevated anti-PNS antibody levels were detected in 4 of 98 (4.1%) leprosy patients (4 of 52, 7.7%, lepromatous leprosy patients), in 1 of 28 (3.6%) contacts, and in 1 of 18 (5.6%) normal controls. There was no correlation between anti-PNS antibody levels and the bacterial index or neuropathy in leprosy. Immunoblot with a sample of six leprosy and five control sera showed that the antigenic binding pattern (mainly within the 100-200-kDa region) was very similar in patients and controls. Staining intensity, however, appeared to be higher with the leprosy sera than with the control sera. IgM and IgG were found to contribute to the staining pattern: IgM in the 150-200-kDa range, IgG with multiple bands between 25 kDa and 200 kDa. Thus, the presence and levels of serum anti-PNS antibodies in leprosy appear to be unrelated to parameters of disease activity, neuropathy in particular, and do not seem to be critically involved in the pathogenesis of nerve damage.

Serum IgA1 and IgM antibodies against Mycobacterium leprae-derived phenolic glycolipid-I: a comparative study in leprosy patients and their contacts.

In order to evaluate the potentials of IgA1 versus IgM as well as of native phenolic glycolipid-I (PGL-I) versus PGL-I-disaccharide coupled to bovine serum albumin (D-BSA) as antigens in the serodiagnosis of leprosy, anti-D-BSA IgA1 and anti-PGL-I IgM were investigated and compared to anti-PGL-I IgA1 in sera from patients and contacts. Anti-D-BSA and anti-PGL-I IgA1 significantly correlate in patients and contacts. The higher IgA1 positivity rates obtained with D-BSA as compared to PGL-I may suggest D-BSA as the favorable antigenic material. In patients but not in contacts anti-PGL-I IgM and IgA1 correlate, IgM predominating over IgA1. In all three antibody systems, the mean values as well as the positivity rates increased from the tuberculoid toward the lepromatous disease pole. Also, the levels of all three antibodies significantly increased with the bacterial index (BI). However, anti-D-BSA (PGL-I) IgA1 appears to be preferable to IgM with respect to sensitivity, i.e., detection of disease activity, in paucibacillary or BI-negative patients. A number of contacts were detected as seropositive with anti-D-BSA and/or anti-PGL-I IgA1 but not with anti-PGL-I IgM. This suggests that IgA1 is a better tool than IgM for the detection of leprosy in its subclinical stage.

IgA antibodies against phenolic glycolipid I from Mycobacterium leprae in serum of leprosy patients and contacts: subclass distribution and relation to disease activity.

The anti-PGL-I IgA response against phenolic glycolipid I (PGL-I) a specific surface antigen of Mycobacterium leprae, was demonstrated to be essentially of the IgA1 subclass in sera from leprosy patients and contacts. Anti-PGL-I IgA1 mean levels were found to increase significantly from the tuberculoid toward the lepromatous pole of the leprosy disease spectrum, thus resembling the predominating anti-PGL-I IgM response. Furthermore, anti-PGL-I IgA1 values were shown to increase significantly with increasing bacillary load, measured as bacillary index (BI) from skin biopsies. However, a number of BI negative leprosy patients recorded elevated anti-PGL-I IgA1 levels possibly reflecting a persistence of disease activity. Three of 28 household or family contacts of leprosy patients were detected seropositive for anti-PGL-I IgA1. Thus, our results suggest that anti-PGL-I IgA1 may be considered as an additional parameter for the early detection of infection with M. leprae.