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Use of reconstructed epidermis to assess keratinocyte activation by skin irritant and sensitizing compounds
ESDR I JSID I SID Abstracts
1108
1105 EXPRESSION
INTERLEUKW-15
USE OF RECONSTRUCTED EPIDERMIS TO ASSESS KERATWOCYTE ACTTATION BY SKIN IRRITANT ,AND SENSTFG COMPOUNDS. N_ &ma. A. Cog&@?. A. Va&ntnxch Y PO _ Department of Histology, University of Namer, Namur and ‘Bio-Pba&a S.I., Wavrc, Belgium. A model of reconsrmctcd epidermis (Rep) obtained from Skinethic was used as an in vitro skin model to discriminate the effect of know” primary contact trritaot and sensitizing compounds. The Reps were topically exposed for 20b (0.125-4 mg/ml) to three irritating (hcozalkonium chloride (BZK), Trite” Xl00 and Twccn 80) and one sensitizing (dinttrochlorobcozcne (DNCB)) compounds. These moIccuIcs produced different dose-dcpcndcnt dccrcasc in celI viability, corresponding with their in viva irritative potency: BZK > DNCB > Triton > Tween. Using morphological observations, Triton Xl00 and DNCB at 2 mg/ml were found to damage the Rep, leading to a disaggregatcd comified layer. Tbe release of intcrleokin-1 alpha (IL-la), intcrlcukin-8 (IL-S) and tum”“~ “ccrosts factor alpha (TNF-a) in the extmccllular medium was measured by ELISA. The rclcasc of IL-la was clearly dose-dependent and correlated with the potency of the irritant compounds. lo idenucal conditions, no significant increase in IL-8 and TNF-a was observed. On the opposite, DNCB did not induce aov sieniticaot increase in &la release. but induced the rclcasc of IL-8 and TNF-a. ti$?R analysis of the expression of these cytokines showed an increase of their corrcswxling mRNA at mild concentrations, but then revealed a denease at the highest co&“trations tested in the assay. These results could be explained by the loss of tissue viability at the highest concentrations aftcr tbc 20h treatment. Our results demonstrate that this in vitro ski” model is a powerful tool for in vitro skin toxicological investigations and suggest that the production of cytokines could bc related to the type of applied compound, whether irritant or sensitizing.
HUMAN
BY
France. loterleokio-15 (IL-15) is a 14-15 kDa cytokine of the fouralpha-helix bundle family which shares many biological activities with IL-2 both support proliferation of T cells and enhance the cytolytic fonctioos of CDE+ T cells and NK cells. Unlike L-2, IL-15 mRNA have been identified in a variety of tissues and cell lines. lo this study, by in sit” hybridization, we have show” that human keratioocytcs isolated from normal skin clearly exprcsscd IL-15 transcripts. In contrast, immonofloorcscencc studies showed a very low expression of U-15 protein by these same cells. This opposition between IL-15 mRNA expression and IL15 protein expression was also observed in vivo by in sit” bvbtidizatio” and immunobistochcmistw on cutane”“s sections p&pared from normal human ski” biop&s. Interestingly, these studies also showed a large increase of IL15 protein &@ssion by c~idcrmal keratinocyies of patients with cutaneous T cell lympboma. whether a correlation exists between tbis level of keratioocytc IL-15 expression and stage of the disease “I degree of disease’s remission under treatment by alpha-interferon therapy remains to bc dctcrmiocd.
1109
1106 A SUBSTANCE
P AGONlST
HAPlET-SPlIClFIC Wavnc
S@eiIein*. ‘Department Japan,
Medical
application,
(CH) induction
exposed
and prevented
that SP agonist enhanced
the gcncration
“or
intrademxally,
doses of haptcn.
at Tokyo,
of Dcnnatology,
from cutaoeous
sensory in contact
radiation
Moreover,
Lagerbans
immooogenic
that promotes
the delctcrious
effects of WR
injcctcd SP cells, implying signals from the of CU
CH and tolemocc
may rcoder the
on cutaocoos
ATOPIC DtRMArITISA AND LATEX ALLERGY, Ikczawa’. Hmxh, Osooa’, Mibo Yamamoto’. Mmwa Okaoma’. SUM Onuma’. luoko Osawal. Kazuko Kttamura’. Kazufum, Tsubaki’aod Tak& Y.&rm7. Department of Dermatology, Yokohama at, Univeraty .Schml of Me&me llralune Hospital, Yokohama,‘Allergcn-l’rce Technology Lab Inc. ‘Nat,ooal Institute of Hcaltb Sc~enccs, Tokyo, Japan Atopic deonah~s(AD) IS well known as ow of scvcral nsk factors m development of latex allergy. but the patbomechan~sm and factors mvolvcd rcmaio unclear In thx paper. the freaueocv of I& anubod~s to latex and other olant allowens and the cros-reacoons were exami&by bog the sem lrom 113 .AD patie’nts Aka.& responses to latex by histamine release test(HRT) and by skm prick tcsting(SET) in the 10 patleota wltb latex-r&cd contact allergy syndromes(CASs) were examined m comparison with those in 13 patients wItbout CASs ‘The frequency of posnivc latex-RAST was 41% m 4D paticol, while 0% io healthy controls The s,ga,f,cant corrclauon were observed not only between latex and fro& amoog latex, vegetables sod cereals The IgI-ELIS.4 men to latex were mhlbtted m adosedependent maooer by addition of banana. avocado. bamboo shoot. spmach and foxlad rmllct extracts. A measurement on I&-bmdmg activity of latex-derived defence related pmtcms (DRS) to the latex-RASI positwe scm from .AD patxnts revealed that some latex-RAST paltlW sera fmnl AD pauents reacted With fl-1.3.glucanase and estcraselprotcasc of DRF from the lax These determmaou on the DRs have been detected as crm-reactmg plant panallergeos mother many planu beyond the phytogenetxally classes of plant Thence. these protans are presumed to be mvolved LOthe mducuon of aoo-latex IgE anhbcdy wtb a tugb frequency in AD patients. and then may result m a high conelatloo cozffuzientbetween IgE antibody levels to latex a,xl otbcr many plant allergens. The percentage of posmve cases by RAST. HRT aml SPI m the patients with CASs was 80% 80% and 80%. respechvely, whde that m the patmnts without CASs was 92%. 0% and 5%. respectively Furthermore. the SIT and HRT wth the sbwr-meotioncd DRs m AD patients with latex allergy showed a posltrve response Both of HRT sod SPf arc usefulness tbr diagoosis of latex allergy, and these DRs are presumed to bc owolved in latex allergy of some casa as active allergen molecules
butalso
on skin
the induction
impaired
of native SP to local ncompeptidases
[Pro’, CH
SP agonist
was painted
(UVR). Intradermally
of hapten-specific
nave
or%Aminovaleryl
SP agonist cohanccd
exogenous SP agonist can prevent
unable to prevent
and J.
SP participates
when hapten
that SP is a natural “adjuvant”
skin. Although
after LJVR, the susceptibility neompcptide
whether
morphology of cpidcrmaI
density
It is proposed
to be released
tolerance
to acute, low dose ultraviolet-B
within non&
and Department
by using SP agonist.GR73632
agonist altercdncithcrtbe dermis.
The Second National Hospital
WC determined
but not optimal,
CH induction
Iwao Kuromoto2
MA, USA.
P,_,,. When injected
by convcntio”aJ,
Leoro
TO PROMOTE
Niizcki’.
Institute
P (SP) has been rcpottcd
N-Me-Leu’O]-substance induced
Hiinori
Eye Research
School, Boston,
endings after bapte”
promoted
AS AN ADJUVANT
of Dermatology,
and 2Scbcpens
Since substance hypersensitivity
ACTS
SKIN IMMUNITY.
Tokyo, Harvard
S185
immunity.
1107
1110
ULTRAVIOLET-B RADIATION AUGMENTS THE INTERLEUKIN-15 mRNA PRODUCTION BY CULTURED NORMAL HUMAN KERATINGCYTES Marcel B.M. Teonissen. Carolvn Foe-A-Man. Cock W. Koomen and Jao D. Bos. Deparrment of Dermatology, Academic Medical Center, University of Amsterdam, Amsterdam. The Netherlands. Normal human keratinocytes (NHK) can produce a wide variety of hmounomodulatory factors and the production of many of these factors have been demonstrated to be affected by ultraviolet-B (UVB) radiation. Interleukin-15 (K-15) is a recently described cytokine, which possess IL-2-like activity, such as stimulation of T cell proliferation. mere are two IL-15 mRNA isofortns, having a different length of the leader peptide region but both leading to an identical mature a-15 protein. NHK arc able to express IL-15 mRNA. Surprisingly, two contradictory publications appeared on the effect of UVB on the IL-15 production by NHK: as monitored by semiquantitative RT-PCR, UVB was reported to cause upregulation and down-regulation, respectively. In this study we exposed cultured NHK to graded doses of UVB (up to 200 J/n?) and isolated the RNA at various timepaints post-irradiation. To examine the presence of IL-15 mRNA splice variants we used RT-PCR. The Northern blot tccbnique was applied to detect the presence of mRNA for IL-15 in a quantitative way, using a radiolabeled IL-15 probe and a phosphor-imager to quantify the IL-15-specific signal. We found that both IL-15 mRNA isofonns were present in NHK. Tbe Northern blots showed that NHK have a constitutive production of IL-15 mRNA. Upon UVB exposure the expression of IL-15 mRNA remained constant or was slightly augmented in a dose-dependent fashion. Presence of IL-8 mRNA in NHK, included as control for adwate UVB stimulation, was clearly increased by UVB Although our results indicate that UVB stimulates the IL-15 mRNA expression, it is important to know whether this will ultimately lead to increased IL-15 protein production.
THE INHIBITORY EFFECT OF ANTI-IL-6 ON EOSINOPHIL. INFILTRATION TO L4MINA PROPRIA INDUCED BY ORAL ADMINISTRATION OF OVALBlJMIN. Sarmiae Bae. Junicbi Hakucawa, Yoicbi Tanaka and Icbim Katavama, Department of Dermatology, Nagasaki University School of Medicine,, Narrasaki. Jaoan. &ad k considered to be an important allergen in induction and progression of atopic dermatitis. In t&d allergy. the eoainopbil is recognimd to be an important effector cell to induce allergic reaction. IL6 baa been reported to be B strong differentiation and chemotaetic factor for eoaiwpbil. The purpose of this study is to elucidate the mle of TbZ eytokine, intsrleukin8, for the eosinopbil infiltration to the lamina propria in the mice sanaitti intraperitn”eaIly and challenge orally with ovalbumin (OVA). For nennitizatioq Balb/c mice were immunized intraoeritineaIlv with 2w OVA and 5 me alum twice on dav 0 and 10. Marked edema OFvilli and &tense e&in”pbil tit&ion to lamina p&pria with peak reeponse at 6br at& challenge was observed a&r oral challenge with OVA on day 20. Expression of IL-6 in the infiltrating cells to the lamina propria WBB demon&rated both at protein and mRNA level by immunobistacbemietry and in-situ hybridization. Furthermore, the intiperitoneal pretreatment of anti-mouse IL6 monoclonal antibody before tbe challenge markedly decreased the eosinopbil i<ration. These resultrr suggest that food allergen can indues allergic reaction in the gut organ and that IL6 plays an important mle on eosinephil infiltration to the lamioa proprie induced by oral challenge with OVA.
1108
1105 EXPRESSION
INTERLEUKW-15
USE OF RECONSTRUCTED EPIDERMIS TO ASSESS KERATWOCYTE ACTTATION BY SKIN IRRITANT ,AND SENSTFG COMPOUNDS. N_ &ma. A. Cog&@?. A. Va&ntnxch Y PO _ Department of Histology, University of Namer, Namur and ‘Bio-Pba&a S.I., Wavrc, Belgium. A model of reconsrmctcd epidermis (Rep) obtained from Skinethic was used as an in vitro skin model to discriminate the effect of know” primary contact trritaot and sensitizing compounds. The Reps were topically exposed for 20b (0.125-4 mg/ml) to three irritating (hcozalkonium chloride (BZK), Trite” Xl00 and Twccn 80) and one sensitizing (dinttrochlorobcozcne (DNCB)) compounds. These moIccuIcs produced different dose-dcpcndcnt dccrcasc in celI viability, corresponding with their in viva irritative potency: BZK > DNCB > Triton > Tween. Using morphological observations, Triton Xl00 and DNCB at 2 mg/ml were found to damage the Rep, leading to a disaggregatcd comified layer. Tbe release of intcrleokin-1 alpha (IL-la), intcrlcukin-8 (IL-S) and tum”“~ “ccrosts factor alpha (TNF-a) in the extmccllular medium was measured by ELISA. The rclcasc of IL-la was clearly dose-dependent and correlated with the potency of the irritant compounds. lo idenucal conditions, no significant increase in IL-8 and TNF-a was observed. On the opposite, DNCB did not induce aov sieniticaot increase in &la release. but induced the rclcasc of IL-8 and TNF-a. ti$?R analysis of the expression of these cytokines showed an increase of their corrcswxling mRNA at mild concentrations, but then revealed a denease at the highest co&“trations tested in the assay. These results could be explained by the loss of tissue viability at the highest concentrations aftcr tbc 20h treatment. Our results demonstrate that this in vitro ski” model is a powerful tool for in vitro skin toxicological investigations and suggest that the production of cytokines could bc related to the type of applied compound, whether irritant or sensitizing.
HUMAN
BY
France. loterleokio-15 (IL-15) is a 14-15 kDa cytokine of the fouralpha-helix bundle family which shares many biological activities with IL-2 both support proliferation of T cells and enhance the cytolytic fonctioos of CDE+ T cells and NK cells. Unlike L-2, IL-15 mRNA have been identified in a variety of tissues and cell lines. lo this study, by in sit” hybridization, we have show” that human keratioocytcs isolated from normal skin clearly exprcsscd IL-15 transcripts. In contrast, immonofloorcscencc studies showed a very low expression of U-15 protein by these same cells. This opposition between IL-15 mRNA expression and IL15 protein expression was also observed in vivo by in sit” bvbtidizatio” and immunobistochcmistw on cutane”“s sections p&pared from normal human ski” biop&s. Interestingly, these studies also showed a large increase of IL15 protein &@ssion by c~idcrmal keratinocyies of patients with cutaneous T cell lympboma. whether a correlation exists between tbis level of keratioocytc IL-15 expression and stage of the disease “I degree of disease’s remission under treatment by alpha-interferon therapy remains to bc dctcrmiocd.
1109
1106 A SUBSTANCE
P AGONlST
HAPlET-SPlIClFIC Wavnc
S@eiIein*. ‘Department Japan,
Medical
application,
(CH) induction
exposed
and prevented
that SP agonist enhanced
the gcncration
“or
intrademxally,
doses of haptcn.
at Tokyo,
of Dcnnatology,
from cutaoeous
sensory in contact
radiation
Moreover,
Lagerbans
immooogenic
that promotes
the delctcrious
effects of WR
injcctcd SP cells, implying signals from the of CU
CH and tolemocc
may rcoder the
on cutaocoos
ATOPIC DtRMArITISA AND LATEX ALLERGY, Ikczawa’. Hmxh, Osooa’, Mibo Yamamoto’. Mmwa Okaoma’. SUM Onuma’. luoko Osawal. Kazuko Kttamura’. Kazufum, Tsubaki’aod Tak& Y.&rm7. Department of Dermatology, Yokohama at, Univeraty .Schml of Me&me llralune Hospital, Yokohama,‘Allergcn-l’rce Technology Lab Inc. ‘Nat,ooal Institute of Hcaltb Sc~enccs, Tokyo, Japan Atopic deonah~s(AD) IS well known as ow of scvcral nsk factors m development of latex allergy. but the patbomechan~sm and factors mvolvcd rcmaio unclear In thx paper. the freaueocv of I& anubod~s to latex and other olant allowens and the cros-reacoons were exami&by bog the sem lrom 113 .AD patie’nts Aka.& responses to latex by histamine release test(HRT) and by skm prick tcsting(SET) in the 10 patleota wltb latex-r&cd contact allergy syndromes(CASs) were examined m comparison with those in 13 patients wItbout CASs ‘The frequency of posnivc latex-RAST was 41% m 4D paticol, while 0% io healthy controls The s,ga,f,cant corrclauon were observed not only between latex and fro& amoog latex, vegetables sod cereals The IgI-ELIS.4 men to latex were mhlbtted m adosedependent maooer by addition of banana. avocado. bamboo shoot. spmach and foxlad rmllct extracts. A measurement on I&-bmdmg activity of latex-derived defence related pmtcms (DRS) to the latex-RASI positwe scm from .AD patxnts revealed that some latex-RAST paltlW sera fmnl AD pauents reacted With fl-1.3.glucanase and estcraselprotcasc of DRF from the lax These determmaou on the DRs have been detected as crm-reactmg plant panallergeos mother many planu beyond the phytogenetxally classes of plant Thence. these protans are presumed to be mvolved LOthe mducuon of aoo-latex IgE anhbcdy wtb a tugb frequency in AD patients. and then may result m a high conelatloo cozffuzientbetween IgE antibody levels to latex a,xl otbcr many plant allergens. The percentage of posmve cases by RAST. HRT aml SPI m the patients with CASs was 80% 80% and 80%. respechvely, whde that m the patmnts without CASs was 92%. 0% and 5%. respectively Furthermore. the SIT and HRT wth the sbwr-meotioncd DRs m AD patients with latex allergy showed a posltrve response Both of HRT sod SPf arc usefulness tbr diagoosis of latex allergy, and these DRs are presumed to bc owolved in latex allergy of some casa as active allergen molecules
butalso
on skin
the induction
impaired
of native SP to local ncompeptidases
[Pro’, CH
SP agonist
was painted
(UVR). Intradermally
of hapten-specific
nave
or%Aminovaleryl
SP agonist cohanccd
exogenous SP agonist can prevent
unable to prevent
and J.
SP participates
when hapten
that SP is a natural “adjuvant”
skin. Although
after LJVR, the susceptibility neompcptide
whether
morphology of cpidcrmaI
density
It is proposed
to be released
tolerance
to acute, low dose ultraviolet-B
within non&
and Department
by using SP agonist.GR73632
agonist altercdncithcrtbe dermis.
The Second National Hospital
WC determined
but not optimal,
CH induction
Iwao Kuromoto2
MA, USA.
P,_,,. When injected
by convcntio”aJ,
Leoro
TO PROMOTE
Niizcki’.
Institute
P (SP) has been rcpottcd
N-Me-Leu’O]-substance induced
Hiinori
Eye Research
School, Boston,
endings after bapte”
promoted
AS AN ADJUVANT
of Dermatology,
and 2Scbcpens
Since substance hypersensitivity
ACTS
SKIN IMMUNITY.
Tokyo, Harvard
S185
immunity.
1107
1110
ULTRAVIOLET-B RADIATION AUGMENTS THE INTERLEUKIN-15 mRNA PRODUCTION BY CULTURED NORMAL HUMAN KERATINGCYTES Marcel B.M. Teonissen. Carolvn Foe-A-Man. Cock W. Koomen and Jao D. Bos. Deparrment of Dermatology, Academic Medical Center, University of Amsterdam, Amsterdam. The Netherlands. Normal human keratinocytes (NHK) can produce a wide variety of hmounomodulatory factors and the production of many of these factors have been demonstrated to be affected by ultraviolet-B (UVB) radiation. Interleukin-15 (K-15) is a recently described cytokine, which possess IL-2-like activity, such as stimulation of T cell proliferation. mere are two IL-15 mRNA isofortns, having a different length of the leader peptide region but both leading to an identical mature a-15 protein. NHK arc able to express IL-15 mRNA. Surprisingly, two contradictory publications appeared on the effect of UVB on the IL-15 production by NHK: as monitored by semiquantitative RT-PCR, UVB was reported to cause upregulation and down-regulation, respectively. In this study we exposed cultured NHK to graded doses of UVB (up to 200 J/n?) and isolated the RNA at various timepaints post-irradiation. To examine the presence of IL-15 mRNA splice variants we used RT-PCR. The Northern blot tccbnique was applied to detect the presence of mRNA for IL-15 in a quantitative way, using a radiolabeled IL-15 probe and a phosphor-imager to quantify the IL-15-specific signal. We found that both IL-15 mRNA isofonns were present in NHK. Tbe Northern blots showed that NHK have a constitutive production of IL-15 mRNA. Upon UVB exposure the expression of IL-15 mRNA remained constant or was slightly augmented in a dose-dependent fashion. Presence of IL-8 mRNA in NHK, included as control for adwate UVB stimulation, was clearly increased by UVB Although our results indicate that UVB stimulates the IL-15 mRNA expression, it is important to know whether this will ultimately lead to increased IL-15 protein production.
THE INHIBITORY EFFECT OF ANTI-IL-6 ON EOSINOPHIL. INFILTRATION TO L4MINA PROPRIA INDUCED BY ORAL ADMINISTRATION OF OVALBlJMIN. Sarmiae Bae. Junicbi Hakucawa, Yoicbi Tanaka and Icbim Katavama, Department of Dermatology, Nagasaki University School of Medicine,, Narrasaki. Jaoan. &ad k considered to be an important allergen in induction and progression of atopic dermatitis. In t&d allergy. the eoainopbil is recognimd to be an important effector cell to induce allergic reaction. IL6 baa been reported to be B strong differentiation and chemotaetic factor for eoaiwpbil. The purpose of this study is to elucidate the mle of TbZ eytokine, intsrleukin8, for the eosinopbil infiltration to the lamina propria in the mice sanaitti intraperitn”eaIly and challenge orally with ovalbumin (OVA). For nennitizatioq Balb/c mice were immunized intraoeritineaIlv with 2w OVA and 5 me alum twice on dav 0 and 10. Marked edema OFvilli and &tense e&in”pbil tit&ion to lamina p&pria with peak reeponse at 6br at& challenge was observed a&r oral challenge with OVA on day 20. Expression of IL-6 in the infiltrating cells to the lamina propria WBB demon&rated both at protein and mRNA level by immunobistacbemietry and in-situ hybridization. Furthermore, the intiperitoneal pretreatment of anti-mouse IL6 monoclonal antibody before tbe challenge markedly decreased the eosinopbil i<ration. These resultrr suggest that food allergen can indues allergic reaction in the gut organ and that IL6 plays an important mle on eosinephil infiltration to the lamioa proprie induced by oral challenge with OVA.