Biomedical subjects

W S Koh

Publications and source records attributed to W S Koh.

At least 19 recordsLinked to original sources

PubMed2001

Evaluation of developmental toxicity in rats exposed to the environmental estrogen bisphenol A during pregnancy.

Bisphenol A (BPA) is an essential component of epoxy resins used in the lacquer lining of metal food cans, as a component of polycarbonates, and in dental sealants. The present study was conducted in an attempt to evaluate the adverse effects of the environmental estrogen BPA on initiation and maintenance of pregnancy and embryofetal development after maternal exposure during the entire period of pregnancy in Sprague-Dawley rats. The test chemical was administered by gavage to mated females from days 1 to 20 of gestation (sperm in varginal lavage = day 0) at dose levels of 0, 100, 300, and 1000 mg/kg. All females were subjected to caesarean section on day 21 of gestation and their fetuses were examined for external, visceral and skeletal abnormalities. In the 1000 mg/kg group, significant toxic effects including abnormal clinical signs, decreased maternal body weight and body weight gain, and reduced food consumption were observed in pregnant rats. An increase in pregnancy failure was also found in the successfully mated females. In addition, increased number of embryonal deaths, increased postimplantation loss, reduced litter size and fetal body weight, and decreased number of fetal ossification centers of several skeletal districts were seen. On the contrary, no significant changes induced by BPA were detected in the number of corpora lutea and implantation sites and by fetal morphological examinations. In the 300 mg/kg group, suppressed maternal body weight and body weight gain, decreased food intake and reduced body weight of male fetuses were seen. There were no adverse signs of either maternal toxicity or developmental toxicity in the 100 mg/kg group. It was concluded that BPA administration during the entire period of pregnancy in rats produced pregnancy failure, pre- and postimplantation loss, fetal developmental delay and severe maternal toxicity, but no embryo-fetal dysmorphogenesis at an oral exposure level of 1000 mg/kg.

Air Pollutants↗

PubMed2001

Role of corticosterone in ethyl carbamate-induced immunosuppression in female BALB/c mice.

We have recently demonstrated that the antibody response to the T-cell-dependent antigen, sheep red blood cells (SRBCs), was suppressed by ethyl carbamate in female BALB/c mice. At the same doses, ethyl carbamate decreased in the numbers of splenic macrophages, B cells, total T cells, CD4(+) T cells and CD8(+) T cells. In addition, the serum level of corticosterone was increased dose-dependently. To investigate the possible role of corticosterone in ethyl carbamate-induced immunosuppression, the antibody response to SRBCs and the subpopulation changes of splenocytes and thymocytes were determined in naive, sham-operated and adrenalectomized (ADX) female BALB/c mice. When the mice were treated intraperitoneally with 400 mg/kg ethyl carbamate, the antibody response was significantly suppressed by ethyl carbamate in naive and sham-operated mice in accompanying the decrease in spleen and thymus weights and/or the increase in the level of serum corticosterone. Meanwhile, the antibody response was not suppressed by ethyl carbamate in the ADX mice. The splenic numbers of total cells, macrophages, B and T cells, and CD4(+) cells were decreased by ethyl carbamate in naive and sham-operated mice. Meanwhile, each cell number was comparable with control in the ADX mice. The flow cytometric analyses on thymocytes did not show obvious differences as seen in the spleen. Finally, when the ADX mice were treated intraperitoneally with 25 mg/kg corticosterone, the antibody response was significantly suppressed. Taken together, our present results suggested that corticosterone might be, at least partially, responsible for ethyl carbamate-induced immunosuppression in female BALB/c mice.

Adrenalectomy↗

PubMed2000

Elevation of the gamma-aminobutyric acid transaminase expression in the gerbil CA1 area after ischemia-reperfusion damage.

gamma-Aminobutyric acid-transaminase (GABA-T) plays an important role in the metabolism of GABA, particularly in the neurons or glial cells. The present study was undertaken to determine the alteration of GABA-T expression in the gerbil hippocampus after ischemia-reperfusion. In the sham, GABA-T(+) neurons were scattered in the hippocampus proper and dentate gyrus. The intensity of the GABA-T immunoreactivity had nearly disappeared in the interneurons at 12 h after ischemia. In contrast, 24 h post-ischemia the dramatic augmentation of GABA-T immunoreactivity in the pyramidal cells was observed in the CA1 area but not in the CA2 or CA3 areas. Forty-eight hours after ischemia-reperfusion, its immunoreactivity was preserved in the CA1 neurons. These results suggest that the over-expression of GABA-T in the CA1 area may be related to delayed neuronal death after ischemia-reperfusion insult.

4-Aminobutyrate Transaminase↗

PubMed1999

Cloning and functional characterization of the human sodium-dependent vitamin C transporters hSVCT1 and hSVCT2.

Two sodium-dependent vitamin C transporters, hSVCT1 and hSVCT2, were cloned from a human kidney cDNA library. hSVCT1 had a 1797 bp open reading frame encoding a 598 amino acid polypeptide. The 1953 bp open reading frame of hSVCT2 encoded a 650 amino acid polypeptide. Using a Xenopus laevis oocyte expression system, both transporters were functionally expressed. By Eadie-Hofstee transformation the apparent K(m) of hSVCT1 for ascorbate was 252.0 microM and of hSVCT2 for ascorbate was 21.3 microM. Both transporters were sodium-dependent and did not transport dehydroascorbic acid. Incubation of oocytes expressing either transporter with phorbol 12-myristate 13-acetate (PMA) inhibited ascorbate transport activity. Availability of the human transporter clones may facilitate new strategies for determining vitamin C intake.

Amino Acid Sequence↗

PubMed1999

Acetylaminofluorene inhibits nitric oxide production in LPS-stimulated RAW 264.7 cells by blocking NF-kappa B/Rel activation.

The mechanism by which 2-acetylaminofluorene (AAF) inhibited nitric oxide (NO) formation, in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was investigated. The decrease in NO, as demonstrated by measurement of nitrite was found to correlate well with a decrease in inducible nitric oxide synthase (iNOS) mRNA. Since the promoter in iNOS gene contains binding motifs for NF-kappa B/Rel, AP-1, and NF-IL6 which appear to be important for LPS-mediated iNOS induction, the effect of AAF on the activation of these transcription factors was determined. Treatment of AAF to RAW 264.7 cells induced a dose-related inhibition of NF-kappa B/Rel in chloramphenicol acetyltransferase activity, while either AP-1 or NF-IL6 activation was not affected by AAF. Treatment of RAW 264.7 cells with AAF inhibited protein/DNA binding of NF-kappa B/Rel to its cognate site as measured by electrophoretic mobility shift assay. In addition, AAF treatment caused a significant reduction of nuclear c-rel, p65, and p50 protein levels, and this decrease was paralleled by the accumulation of cytoplasmic c-rel, p65, and p50. These data suggest that AAF inhibits iNOS gene expression by a mechanism involving a blockade of LPS-induced nuclear translocation of NF-kappa B/Rel.

2-Acetylaminofluorene↗

PubMed1998

Inhibition of the cyclic AMP signaling cascade and nuclear factor binding to CRE and kappaB elements by cannabinol, a minimally CNS-active cannabinoid.

Immune suppression by cannabinoids has been widely demonstrated in a variety of experimental models. The identification of two major types of G-protein-coupled cannabinoid receptors expressed on leukocytes, CB1 and CB2, has provided a putative mechanism of action for immune modulation by cannabinoid compounds. Ligand binding to both receptors negatively regulates adenylate cyclase, thereby lowering intracellular cyclic AMP (cAMP) levels. In the present studies, we demonstrated that cannabinol (CBN), a ligand that exhibits higher binding affinity for CB2, modulates immune responses and cAMP-mediated signal transduction in mouse lymphoid cells. Direct addition of CBN to naive cultured splenocytes produced a concentration-dependent inhibition of lymphoproliferative responses to anti-CD3, lipopolysaccharide, and phorbol-12-myristate-13-acetate/ionomycin stimulation. Similarly, a concentration-related inhibition of the in vitro anti-sheep red blood cell IgM antibody forming cell response was also observed by CBN. Evaluation of cAMP signaling in the presence of CBN showed a rapid and concentration-related inhibition of adenylate cyclase activity in both splenocytes and thymocytes. This decrease in intracellular cAMP levels produced by CBN resulted in a reduction of protein kinase A activity, consequently leading to an inhibition of transcription factor binding to the cAMP response element and kappaB motifs in both cell preparations. Collectively, these results demonstrate that CBN, a cannabinoid with minimal CNS activity, inhibited both cAMP signal transduction and immune function, further supporting the involvement of CB2 receptors in immune modulation by cannabimimetic agents.

Activating Transcription Factor 2↗

PubMed1998

Cinnamaldehyde inhibits lymphocyte proliferation and modulates T-cell differentiation.

Two kinds of cinnamaldehyde derivative, 2'-hydroxycinnamaldehyde (HCA) and 2'-benzoxy-cinnamaldehyde (BCA), were studied for their immunomodulatory effects. These compounds were screened as anticancer drug candidates from stem bark of Cinnamomum cassia for their inhibitory effect on farnesyl protein transferase activity. Ras activation, which is accompanied with its farnesylation, has been known to be important in immune cell activation as well as in carcinogenesis. Treatment of these cinnamaldehydes to mouse splenocyte cultures induced suppression of lymphoproliferation following both Con A and LPS stimulation in a dose-dependent manner. A dose of I microM of HCA and BCA inhibited the Con A-stimulated proliferation by 69% and 60%, and the LPS-induced proliferation by 29% and 21%, respectively. However, the proliferation induced by PMA plus ionomycin was affected by neither HCA nor BCA treatment. Decreased levels of antibody production by HCA or BCA treatment were observed in both SRBC-immunized mice and LPS-stimulated splenocyte cultures. The exposure of thymocytes to HCA or BCA for 48 h accelerated T-cell differentiation from CD4 and CD8 double positive cells to CD4 or CD8 single positive cells. The inhibitory effect of cinnamaldehyde on lymphoproliferation was specific to the early phase of cell activation, showing the strongest inhibition of Con A- or LPS-stimulated proliferation when added concomitantly with the mitogens. In addition, the treatment of HCA and BCA to splenocyte cultures attenuated the Con A-triggered progression of cell cycle at G1 phase with no inhibition of S to G2/M phase transition. Although cinnamaldehyde treatment had no effect on the IL-2 production by splenocyte cultures stimulated with Con A, it inhibited markedly and dose-dependently the expression of IL-2Ralpha and interferon-gamma. Taken together, the results in this study suggest both HCA and BCA inhibit the lymphoproliferation and induce a T-cell differentiation through the blockade of early steps in signaling pathway leading to cell growth.

Adjuvants, Immunologic↗

PubMed1997

A screening method of SH2 domain ligands and blockers using a solid phase binding.

We have developed a high throughput screening method for SH2 domain binding ligands and blockers. This method measures directly the binding of a 3H-labeled phosphopeptide derived from the sequence around tyrosine317 in the human Shc (SpYVNVK) to the SH2 domain of Grb2, which is precoated as glutathione S-transferase fusion proteins on solid phase. The optimum concentration for the fusion protein coating was 300 ng/100 microl/well for SH2 domain binding. Although an 8-h incubation at 4 degrees C for the coating of fusion protein was required to reach a maximum binding, even a 2-h coating produced 84% of the maximum binding. Saturation of ligand peptide binding in our assay system was observed at 10 pmol/well for the SH2 domain. However, 2 pmol/well showed consistent and reproducible results for the binding when the incubations were performed for 8 h at 4 degrees C. Competitive binding inhibition studies with various unlabeled phosphopeptides imply that the binding assay is highly specific to peptide sequences and able to screen possible ligands or blockers of signal transduction pathway mediated by Grb2 SH2 binding. In conclusion, our new method for SH2 domain binding is easy, rapid, and most of all inexpensive. These advantages over existing assay methods make this method especially suitable for a high throughput application, such as the screening for anticancer drug candidates.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Dynamin II associates with Grb2 SH3 domain in Ras transformed NIH3T3 cells.

Grb2, a linker protein containing two SH3 domains and one SH2 domain, is known as an essential element of the Ras pathway in multiple systems. One of the functions of Grb2 is to link tyrosine-phosphorylated receptors to downstream effector proteins via the SH2 and SH3 domain bindings. To identify Grb2-associated proteins in Ras transformed NIH3T3 cells, we performed coprecipitation experiments using recombinant GST-Grb2 fusion proteins and found a remarkably strong band of 100 kDa. With N-terminal amino acid sequencing, we identified the protein of 100 kDa as dynamin II. Dynamin II was also observed in the coprecipitates with the GST fusion protein of N-SH3 or C-SH3 domain of Grb2 but not in that of Grb2 SH2 domain. The SH3-mediated association of Grb2 with dynamin II was confirmed by competitive binding experiments with oligopeptides whose sequence corresponded to that of SH2 or SH3 binding motif. The dynamin II coprecipitation was completely abrogated by the addition of the oligopeptide of SH3 binding motif, but addition of SH2 binding motif had no effect. In conclusion, these results suggest that dynamin II may be largely expressed and closely associated with Grb2-mediated signaling in Ras transformed cells.

3T3 Cells↗

PubMed1997

Inhibition of protein kinase A and cyclic AMP response element (CRE)-specific transcription factor binding by delta9-tetrahydrocannabinol (delta9-THC): a putative mechanism of cannabinoid-induced immune modulation.

Delta9-Tetrahydrocannabinol (delta9-THC) binding to cannabinoid receptors induces an inhibition in adenylate cyclase activity through the engagement of a pertussis toxin-sensitive GTP-binding protein. In this study we investigated the ramifications of decreased cyclic AMP (cAMP) formation by delta9-THC on signaling events through the cAMP pathway distal to adenylate cyclase in mouse splenocytes. Delta9-THC treatment produced a marked and concentration-related decrease in forskolin-inducible protein kinase A (PKA) activity. This decrease in kinase activity was due to an inhibition in cAMP formation and not through a direct effect on the kinase as evidenced by the fact that PKA activity could not be modulated directly by delta9-THC in the presence of exogenous cAMP. One of the primary roles of PKA in this signaling pathway is to activate transcription factors for subsequent binding to cAMP response elements (CRE) present in the promoter region of cAMP-responsive genes. In the present studies, we observed that forskolin treatment of splenocytes resulted in a rapid activation of trans-acting factor binding to the CRE, which peaked at 30-60 min and whose binding was repressed concentration dependently in the presence of delta9-THC. As with forskolin, mitogenic stimulation including anti-CD3 mAb or phorbol ester plus ionomycin treatment of splenocytes induced CRE binding activity, which was maximal around 60 min and was suppressed by delta9-THC treatment. In conclusion, these data indicate that cAMP-mediated signal transduction is inhibited by delta9-THC and consequently leads to a decrease in the activation of transcription factors that bind to CRE regulatory sites.

Animals↗

PubMed1997

Transient CRE- and kappa B site-binding is cross-regulated by cAMP-dependent protein kinase and a protein phosphatase in mouse splenocytes.

Cyclic AMP regulates a variety of cellular responses through activation of cAMP-dependent protein kinase (PKA). The catalytic subunit of PKA, in turn, activate cAMP responsive element (CRE) and nuclear factor-kappa B (NF-kappa B) binding proteins. In this study, we demonstrated that binding activity to both CRE and kappa B sites in nuclear extracts from spleen cells is modulated by PKA in a time-dependent manner. Electrophoretic mobility shift assays showed that binding by transcription factors to either the CRE or kappa B motif was rapidly up-regulated by cAMP, with maximum binding detected at 30 min in response to forskolin stimulation of splenocytes. This was followed by a steady decline in CRE and kappa B thereafter reaching basal levels by 2 hr. This up-regulation in CRE and kappa B binding was closely associated with an enhancement of PKA activity which was maximum at 30 min following forskolin stimulation. However, unlike the binding of regulatory factors to CRE and kappa B motifs which was very transient, peak PKA activity was sustained for 2 hr. Interestingly, okadaic acid, a protein phosphatase inhibitor, prevented the decline in protein binding to CRE and kappa B motifs 2 hr following forskolin stimulation and actually produced a slight increase at 30 min. These data suggest that binding by transcription factors to CRE and kappa B sites are up-regulated concomitantly with PKA activation but subsequently down-regulated by a protein phosphatase.

Animals↗

PubMed1997

Fc epsilon RI-ligation induces association of tyrosine phosphorylated proteins with Src homology 2 domains of phospholipase C gamma 1 in RBL-2H3 rat basophilic leukemia cells.

Stimulation of the IgE receptors on mast cells and basophils activates protein tyrosine kinases and phospholipases leading to histamine release. However, the mechanism by which protein tyrosine kinases regulate the phospholipases is not clearly defined yet. In this study, we examined the possibility that phospholipase C gamma 1 associates with protein tyrosine kinases and tyrosine phosphorylated molecules as a result of activation of RBL-2H3 cells, and that this association involves the Src homology 2 domains of phospholipase C gamma 1. An increase in cytoplasmic Ca2+ level and tyrosine phosphorylations of proteins, including 72 and 40 kDa proteins, were observed after the cross-linking of the IgE receptors on RBL-2H3 rat basophilic cells by dinitrophenyl-specific IgE and dinitrophenyl-conjugated human serum albumin. Immunoprecipitation and coprecipitation experiments were performed to determine if the activation of protein tyrosine kinases is linked to the activation of phospholipase C gamma 1 via its SH2 domains. Tyrosine phosphorylation of phospholipase C gamma 1 was observed in 1 min following IgE receptor stimulation. several proteins (72, 50, 40, and 33 kDa) were identified to be tyrosine phosphorylated and specifically associated with phospholipase C gamma 1 by its Src homology 2 domains. In addition, the coprecipitated complex contains the tyrosine kinase activity which phosphorylates 72, 40, and 33 kDa proteins in the complex. In conclusion, these studies establish that tyrosine-phosphorylated proteins of 72, 40, and 33 kDa associate with phospholipase C gamma 1 via its SH2 domains following IgE receptor stimulation of RBL-2H3 basophilic cells, implying that protein tyrosine kinases may tyrosine-phosphorylate and recruit signaling proteins around the phospholipase C gamma 1 and that phospholipase C gamma 1 activation induces calcium mobilization, PKC activation and degranulation in mast cells or basophils.

Animals↗

PubMed1997

Copper vapour laser treatment of port-wine stains in brown skin.

Forty-seven Korean patients with port-wine stains were treated with a copper vapour laser and clinical responses were assessed at three months after the last treatment by comparing photographs taken before each treatment. The immediate histologic changes within 15 min after laser treatment were also observed by routine H&E and nitroblue tetrazolium chloride staining. When we treated port-wine stains with minimal whitening doses of 6-8 J/cm2, no or slight colour changes were obtained. Thus, all port-wine stain lesions in this study were treated with non-specific energy densities ranging from 10-20 J/cm2. Good to excellent results were obtained in 18 (38.2%) of 47 Korean patients with port-wine stains. Repeated treatment can continue to reduce colour. Darker lesions (purple or red) are more likely to result in a marked colour change. At above threshold dose, there was wedge-shaped diffuse coagulation necrosis and loss of viability of the epidermis and underlying dermis. Even though copper vapour laser treatment of port-wine stains in brown skin is not as selective as in white skin because of epidermal melanin, our clinical data demonstrate the usefulness of the copper vapour laser for the treatment of port-wine stains in brown skin.

Adolescent↗

PubMed1996

Cannabinoid inhibition of adenylate cyclase-mediated signal transduction and interleukin 2 (IL-2) expression in the murine T-cell line, EL4.IL-2.

Cannabinoid receptors negatively regulate adenylate cyclase through a pertussis toxin-sensitive GTP-binding protein. In the present studies, signaling via the adenylate cyclase/cAMP pathway was investigated in the murine thymoma-derived T-cell line, EL4.IL-2. Northern analysis of EL4.IL-2 cells identified the presence of 4-kilobase CB2 but not CB1 receptor-subtype mRNA transcripts. Southern analysis of genomic DNA digests for the CB2 receptor demonstrated identical banding patterns for EL4.IL-2 cells and mouse-derived DNA, both of which were dissimilar to DNA isolated from rat. Treatment of EL4.IL-2 cells with either cannabinol or Delta9-THC disrupted the adenylate cyclase signaling cascade by inhibiting forskolin-stimulated cAMP accumulation which consequently led to a decrease in protein kinase A activity and the binding of transcription factors to a CRE consensus sequence. Likewise, an inhibition of phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced interleukin 2 (IL-2) protein secretion, which correlated to decreased IL-2 gene transcription, was induced by both cannabinol and Delta9-THC. Further, cannabinoid treatment also decreased PMA/ionomycin-induced nuclear factor binding to the AP-1 proximal site of the IL-2 promoter. Conversely, forskolin enhanced PMA/ionomycin-induced AP-1 binding. These findings suggest that inhibition of signal transduction via the adenylate cyclase/cAMP pathway induces T-cell dysfunction which leads to a diminution in IL-2 gene transcription.

Adenylyl Cyclase Inhibitors↗

PubMed1996

Histological responses of port wine stains in brown skin after 578 nm copper vapor laser treatment.

BACKGROUND AND OBJECTIVES: The object of this study is to characterize the effects of epidermal melanin in brown skin on selective vessel damage by copper vapor laser radiation in port wine stain (PWS). STUDY DESIGN/MATERIALS, AND METHODS: We observed the histological changes of PWS in Korean patients who received copper vapor laser (578 nm) treatment over a range of energy densities (6-14 J/cm2) and exposure durations (30-200 ms). The nitroblue tetrazolium chloride (NBTC) staining method was used to differentiate between the blue-stained viable cells and the unstained thermally damaged cells. RESULTS: With Fontana-Masson stain, we found that Korean skin has more epidermal melanin than Caucasian skin. For energy densities greater than 6 J/cm2, epidermal damage was observed. At 6 and 8 J/cm2, the damage to the dermis was localized to the blood vessels and the perivascular tissue. The connective tissue between damaged vessels and epidermis was still viable. Energy densities above 10 J/cm2 produced a diffuse thermal necrosis. We conclude that vascular selectivity without epidermal damages cannot be achieved with a 50 ms exposure at 578 nm in the brown skin of Koreans. The energy density for clinical minimal whitening was 6-8 J/cm2, and the maximum penetration depth of these energy densities was 0.4 mm. We also found that the epidermal damage increased with increasing pulse widths at a fixed energy density (10 or 8 J/cm2) while the severity and depth of vascular damage decreased. These findings suggest that it is best to treat PWS with a copper vapor laser at the minimal pulse width and maximal power output possible at given energy density. CONCLUSION: We have demonstrated that the copper vapor laser treatment of PWS in the brown skin is not as selective as in white skin because of epidermal melanin.

Adult↗

PubMed1996

Expression of functional glucagon receptors on lymphoid cells.

The objective of the present studies was to determine whether the existence of functional glucagon receptors could be established on lympoid cells. The glucagon receptor, which positively regulates adenylate cyclase, is a member of the superfamily of seven transmembrane domain G-protein coupled receptors. Previously reported specific binding with [125I]-glucagon to a variety of lymphoid and myeloid cell preparations suggests that glucagon receptors are expressed within the immune system. In the present study, Northern analysis of polyA RNA isolated from primary mouse and rat derived lymphoid tissues and lymphoid cell lines EL-4.IL-2, Jurkat E6-1, CH12LX, and BCL1-3B3 cells were probed with a 32P-labeled human hepatic glucagon receptor. Mouse spleen and thymus, rat spleen, and the B cell line, CH12LX, all possessed a single 1.5 kb fragment (BCL1-3B3, 1.4 kb) which hybridized to the glucagon receptor cDNA probe, as compared to mouse liver which exhibited a 2.8 kb fragment. EL-4.IL-2 and Jurkat E6-1 cells possessed a 3.7 kb fragment with an additional 2.75 kb band present in Jurkat E6-1 cells. Treatment of mouse splenocytes and T- and B-lymphoma cells with glucagon (0 - 100 nM) produced a dose-dependent enhancement in intracellular cAMP which was maximal at 5 min post treatment followed by a gradual decline. Direct addition of glucagon to spleen cell cultures over a broad concentration range produced no effect on either lymphoproliferation following stimulation with anti-CD3 mAb, or LPS nor on the antibody forming cell (AFC) response to sRBC. Conversely, glucagon effectively reversed the suppression of the sRBC AFC response produced by delta9-tetrahydocannabinol (delta9-THC), and partially reversed the suppression produced by 2',3'-dideoxyadenosine, both of which are potent inhibitors of adenylate cyclase. These studies confirm the expression of functional glucagon receptors on lymphoid cells.

Adenylyl Cyclases↗

PubMed1996

Ultraviolet B irradiation-enhanced interleukin (IL)-6 production and mRNA expression are mediated by IL-1 alpha in cultured human keratinocytes.

Ultraviolet (UV) B radiation may trigger cutaneous inflammatory responses by directly inducing epidermal keratinocytes to elaborate specific cytokines such as interleukin (IL-1) and IL-6. Because IL-1 is a potent inducer of IL-6, one may speculate that the release of IL-6 by keratinocytes after UV exposure is mediated via the release of IL-1 in an autocrine or paracrine manner. We demonstrated that UVB irradiation upregulated IL-1 alpha mRNA at a lower dose (15 mJ/cm2) and then downregulated IL-1 alpha mRNA expression at high doses (30-40 mJ/cm2). The kinetic profile of IL-1alpha mRNA expression showed a biphasic response, with the early increase by 1 h after UV exposure and the secondary increase at 6 h after UV. On the other hand, the expression of IL-6 mRNA was increased with increasing doses of UVB (0-45 m/J/cm2) and showed a single peak at 6 h post UV. These results may indicate that UVB radiation could regulate the expression of IL-1alpha and IL-6 mRNA in keratinocytes by different mechanisms. Our data show that anti-human IL-1alpha antibody inhibits UV-induced IL-6 production and mRNA expression in cultured keratinocytes. The addition of recombinant IL-1alpha to the medium increased IL-6 synthesis and augmented IL-6 production and mRNA expression in cultured human keratinocytes by UVB irradiation. These results support the hypothesis that UVB irradiation-enhanced IL-6 production and mRNA expression may be mediated by IL-1alpha.

Animals↗

PubMed1995

Cyclic AMP is an essential factor in immune responses.

In the present studies, cAMP, a secondary messenger historically viewed as a negative mediator of immune responses, was demonstrated to possess immunoenhancing activity at low concentrations. In parallel experiments the adenylate cyclase inhibitor, 2',3'-dideoxyadenosine, produced a marked inhibition of humoral and proliferative immune responses suggesting that cAMP at physiologically relevant concentrations acts as a critical second messenger in immune responses. Direct addition of dibutyryl cAMP (10-100 microM), a membrane permeable cAMP analog, to mouse spleen cell cultures produced a marked and dose-related increase (25-100%) in humoral immune responses as measured by the primary IgM antibody forming cell response to the antigen, sheep erythrocytes. Over a similar concentration range, dibutyryl cAMP (5-50 microM) also dose-dependently enhanced (25-50%) phorbol 12-myristate 13-acetate/ionomycin-stimulated lymphoproliferation. Incubation of spleen cells with 2',3'-dideoxyadenosine (40-80 microM), an adenylate cyclase inhibitor, for 30 min depressed significantly the basal level of intracellular cAMP. 2',3'-dideoxyadenosine treatment also significantly decreased both the antibody forming cell response and the proliferative response in a dose-dependent manner. Interestingly, the antibody forming cell response exhibited significantly greater sensitivity to inhibition by 2',3'-dideoxyadenosine than the lymphoproliferative responses. The critical role for cAMP as a positive immunoregulatory signal is further supported by the fact that the immunosuppression by 2',3'-dideoxyadenosine could be reversed completely in the antibody forming cell response to sheep erythrocytes through the addition of dibutyryl cAMP into the culture medium. Partial but not complete reversal of the inhibitory effects of 2',3'-dideoxyadenosine on lymphoproliferation was also demonstrated by dibutyryl cAMP. Taken together, these results suggest that cAMP acts as a positive regulatory signal for immune responses as indicated by the fact that depletion of intracellular cAMP induces a marked inhibition of humoral and proliferative responses.

Animals↗