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Macrophage colony stimulating factor (M-CSF) protein production by murine but not human keratinocytes
ESDR I JSID I SID Abstracts
1090
1087 METASTATIC
BUT
NEUTROPHIL
ATTACK
INTERLEUKIN-8.
NOT
PRIMARY
INDUCED
Mwhiro
m&ha&,=
CELLS
BY ADENOVIRAI.
ESCAPE
GENE
OF
Satwnoorthv’.
m.
of Dermatology,
FROM
TRANSFER
Ok1’.2a~aettu
and h&e”&&
PA, USA; 2Department
MELANOMA
‘The !%star Kobe University
I”&tute,
(lL-8) IS a chemoattractant and actwator for neut.roph&
uced a recombmant determine
adenovnw
cell bnes in vitro and m viva. IL%Ad5,
IL-8 pro&n
ng/ml/106
cells/48h
affect
contanung
the effect ofoverexpression
their
pnmary
wth
cells
“me.
mhdxted
was
to a lesser
adenowrus-medmted primary
melanoma
possess
a mechantsm
hand,
degree.
of IL-8 has
We hypotbcsrze
for downregulatmn
of
0.19 to 6800 cells dtd not
of IL-8iAd5-m&ted severe
combined
of metastat~cmelnoma
exammatmn
noutrophihc
umtslcell
from
tn
tumorigentaty
to
melanoma
formrng
melanoma
suppressed
Histologxal
prominent
overexpression cells
20 plaque
cells mcreased
We
(lL-S/Ad9
of human
tumongemclty
completely
On the other
cell ~“)ect.lo” showed
Il.-i’ cDNA
by IL-R/Ad&mfected
growth rate m vitro. However,
tmmunodeticient
melanoma
infcxtmn
1” melanoma
IL-8 produced
melanoma
cells was
After
,,roductton
the human
of K-8 on the growth
of the
infiltration.
a potent
that advancedstage
effect
melanoma
of the neutrophd.med~ated
site
of
I” summary,
anti-tumor
OF GENE EXPRESSION
OF PERIPHERAL
BLOGD
Kobe,
Japan Interleolond
ANALYSIS
CELL C!YTOKlNES IN ATOPIC DERMATITIS PATIENTS BY MEANS OF RT-PCR WITH INTERNAL STANDARDS
A. Fujioka, K. Banba, H. Takasu, A. lshibashi, Dept. of Dermatology, National Defense Medical Collcee. T. Toda. Dwf. of Molecular Bioloev. Tokvo Metrocolitan
Pbdadelphia,
School of Mechcine,
QUANTlTATIVE MONONUCLEAR
on
tolls may
of Medicine I” the srudy of mRNA levels of peripheral blood mononuclear cell cytokins in atopic demmtitis o&n&. the conventional RT-PCR method detects the messaees of both ?hl and Th2 cytokines only qualitatively in most cases, without elucidating the immu”“logicai predominance of one over ffie other. In our present study to establish a simple and reliable quant~talive measttrement method, we prepared chimera DNA
.
standards for quantitating IFN- 7 from Thl sod IL-4 from ‘IbZ by amplifying pBR322 DNA using joint primers of human cytokines and the plasmid. The chimera DNAs served as internal standards for PCR. With the mRNA of peripheral blood mo”onuclear cell obtained from atopic detmatifis patients and the healthy control as the mooid, cDNA was synthesized and sobjeued to PCR togethn with the corresponding internal standard, each cytokine placed in one tube. After agamse gel electmphoresis and &idiom bromide staining, the photographic negative picture of tbe specimen was examined by means of densitomcby so as to quantitatively de&nine the expression of cytokins on the basis of their ratio between the internal standard and specimen. As a result, Thl-derived cytokinc was shown to lx more dotizmt in the peripheral blood of patients with chronic atopic dermatitis.
~ytotoxwty.
1088
1091
INCREASED EXPRESSION OF IFN-y IN SMW BOWELBlOPSIES OF PATIENTS WITH SyMpToMAnCGlJJ,‘ENSENSITIVE ENTEROPATHWSE) COMPARED TO PATLENTS
INTERLEUKIN-18 m-18) AND INTERLEUKIN-12 (llx12) SYNERGISTICALLY AUGMENT CELGMEDJA’IED IMMUNITY IN CUTANEOUS T-CELL LYMPHOMA (CZL). EK I&&&i&& Deparnnent of Dermatology, University of Pennsylvania, Philadalnhia PA lmmt&&&&Iities associated with advanced forms of CTCL are chamcteti~d bv marked deficiencies in cell-mediated immwitv and T-heloer tvoe 1 fRt1,
With DERMAnnS HEPzETJFOftMStTXQ. B Bsleti. AD W&_&D Smilien. W Hall. Dt,,h.w,, “A and Duke Medica, Center, Our@ NC “SA. DH patienti have an associated GSE, most oRen wti no ganrcdntcstinaUG1) symptoms, even while on a 8luten contaming diu In mntwt, patients with imiated GSE dwelop Gi symptoms when on a gluten comaining diet hut do twt develop skin disease. We bypothsized daat the diBemt cDnical presematie”s of there loom of GSEarea rewlt of thepatternof cyiekincexpraian in the smallbowel that ecctmwiththe ingestionof@ten Smallbowelbiopsies were obcained from 8 DH patients. alI on gluten contaming diets. 5 padeats with isolated GSE an &ten with xtivs GI symptoms and 4 pauents with is&ted GSE on a @en free diit(GFD) wilhan Gi symptoms. Biopsies were tau, anb mP.NA is&ted. C-DNA was arewed mitt8 reverse trantiowe and PCR tinned with P”. FCR was
&tokirtc production, including interferon gammav(IFN-7). ri-lS:ke k-121 induces IFi+-t production and enhances cytotoxic lymphocyte activity. Since we have show” that IL-12 revetxs the immune defects obsewed in CICL* we investigated the potency of IL-I 8 to synergistically augment IL12 in restoring immune functions in CTCL. Peripheral blood mononocletx cells (PBBMC) from patients with advanced C%!L (n=4) and normal healthy volunteers (“~2) were cultured with medium alone,
IL-18(lOnghnl) or IL-lZ(lng/ml) alone or in combination for 24 hours.
or -12.01 T&a was r&d between pients with DH. symptomatic GSE or asymptomatic GSE Analysis of the IFN-y expression havever reveakd that paoenU with DH an a 8iuten diet had signiticantly lower ratio of IFN+D38 wrpre&n(O.71) than active GSE patienW2 13)( W.05) but r)ot different from asymptomatic patients titb GSE(l.Z2)(pXIO5). Analysis of TGF-B exprcssiott revealed tha active GSE patietientralso had a higher ratio of TGF+/CD3s(4. I) than DH patientsienll(l.22) or asymptamatc GSE patie,,u(O. 18)(p
those patients
in patient PBMC. IL18 alone, however appeared to depress IFN-yproduction in normal PBMC from 658 to 4lOpdt”l and in patient PBMC from 47 to 38pg/ml. When IL-18 was combined with lL-12 a synergistic increase in IFN-yproduction was observed in both normal (1808 to 2338pg/ml) and patient (124 to 165pg/ml) PBMC. Similarly, cultures combining Il.18 and IL12 detnonsuated enhanced NK cell activity in comparison to IL-18 orlL-12 alone. SincelLand IL12 appearto have the capacity to correct some of the immune abnonnalitics associated with advanced Cl’U, our preliminary data have important rhempeutic implications and soppon the rationale fo combine both biologic agents io clinical protocols.
1092
1089 Macrophage Colony Stimulating Factor (M-CSF) human keratinocytes. Martin I.. Johnso~k Cbristoti~ Depts. of DematoI&y
PHA(lfiglml) was then added for the final 24 hours and supernatants wereassayed by ELISA for IFN-7. PBMC were aIs assayed for NK cell activity using cbmmitm51 labeled KS62 cells as targets. When compared to media. IL-12 alone augmented lFN-7 production from 658 to 1808pglml in normal PBMC and from 47 to 124pg/ml
protein production by muine but not Walsh. Katherine A. Kelly, and Immunology, Univ. of Colorado
HSC, Denver. CO, USA; USAFIT, Wright-Patterson AFB. OH, USA; Dept. of Pathology and Immunology, Monash Medical School, Prahmn, Australia. Keratinoeytes actively participate 1” the inklammatory process primarily through the production and secretion of cytokines. One cytokme that has bee” attributed to be part of the cytokine repertoire of mwine and human keratinocytes is macrophage colony stimulating factor or M-CSF. However, previously published data concluding that MCSF is produced by human kerarinocytes is in doubt. By “s~“g Golgi immunostaining, we assessed the expression of M-CSF protein in human primary keratinccytes and the human keratinocyte lines, HaCaT and A431, relative to that in M-CSF-producing celI controls, the nwine keratinocyte lines, PAM212 and XB-2, and the human M-CSFgene transfected murine melanoma Line,B16FlOA2. Intracellular M-CSF protein was identified on an individual cell basis using M-CSF-specific monoclonal antlbodles in pemxablized cells which showed enhanced expression after the addition of monensin for the last 2 hours of culture. M-CSF-producing cells were enumerated by fluorescent microscopy and by stngle-color cytofluommetry. Essentially all of the phorbol acetatestimulated PAM212 control cells and all of the human M-CSF-transfected A2 control cells were M-CSF producers. Smaller numbers of XB-2 cells, control celis that are weak producers of M-CSF, could be. identified as M-CSF producers when these cells were stimulated with phorbol acetate followed by monensin treatment. Enumeration of M-CSF+ cells by flow cytometry, although less sensitive than manual enumerauon of fluorescent cells, and M-CSF bioassays mirrored these findings. However, in contrast to the data of the three different control ceils, M-CSF protein could not be identified in any of the prtmary human keratinocytes “or in the human HaCaT and A43 1 keratinocy~c lines eve” after phorbol acetate stimulation and monensin treatmeot.
~OPHEWTWE CHARA-TloN Gf curANEOIJs lNKrrRATE OF LEsloNAL SKIN BEFORE AND AFTER FXR.4cORFuRF.U PHoIucHEM0-Y IN CTCL PATIENls M. Fimkmi, P. Rubegni, C Miracco’, G. De Aloe, Lm Department of Dermatology, and’ Department of Pathology, University of Siena, Italy. Poticlinim “Lx Scott& 53 100 Siena - ITALY Extracorporeal photochemotherapy release of macrophagedependent currently considered to modulate evaluated the fimction of peripheral
(ECP) has bee” show” to caose proinflammatory cytoldnes and is immune response. I” a previous blood mononuclear cells (PBMC) in
a marked therefore study we 8 patients
with early stage (Ib) c”tnneoos T-4
iymphoma (CTCL). before and one year after ECP. Before treatment. we observed that PBMC aroduced sitmiticantk higher levels of IL4 and lower levels of IF&y t&n in h&thy control subjects.
Afla one year of ECP lI.4 and IFN-y production no longer differed from that of control subjects. We co”clttded that ECP reverses the ThVIh2 imbalance. Here we repoti a retrospective inmunobistochemical study of lesional ski” hefore and after ECP in the same CTCL patients. Immunobistocheutistty wu performed on fured sections using antibodies against CDl% CD3. CD4, CDS. CDS. Mibl, CD30,
CD31, CD45RO and CD45RA by the avidin-biotin complex method. The results showed that ECP is capable 10 modifies the itnmtmopheootype pattern of lesional ski” in6ltrate in early stage CTCL p&nts, pretioosty observed in peripheral blood.
matching
the modifications
that we
1090
1087 METASTATIC
BUT
NEUTROPHIL
ATTACK
INTERLEUKIN-8.
NOT
PRIMARY
INDUCED
Mwhiro
m&ha&,=
CELLS
BY ADENOVIRAI.
ESCAPE
GENE
OF
Satwnoorthv’.
m.
of Dermatology,
FROM
TRANSFER
Ok1’.2a~aettu
and h&e”&&
PA, USA; 2Department
MELANOMA
‘The !%star Kobe University
I”&tute,
(lL-8) IS a chemoattractant and actwator for neut.roph&
uced a recombmant determine
adenovnw
cell bnes in vitro and m viva. IL%Ad5,
IL-8 pro&n
ng/ml/106
cells/48h
affect
contanung
the effect ofoverexpression
their
pnmary
wth
cells
“me.
mhdxted
was
to a lesser
adenowrus-medmted primary
melanoma
possess
a mechantsm
hand,
degree.
of IL-8 has
We hypotbcsrze
for downregulatmn
of
0.19 to 6800 cells dtd not
of IL-8iAd5-m&ted severe
combined
of metastat~cmelnoma
exammatmn
noutrophihc
umtslcell
from
tn
tumorigentaty
to
melanoma
formrng
melanoma
suppressed
Histologxal
prominent
overexpression cells
20 plaque
cells mcreased
We
(lL-S/Ad9
of human
tumongemclty
completely
On the other
cell ~“)ect.lo” showed
Il.-i’ cDNA
by IL-R/Ad&mfected
growth rate m vitro. However,
tmmunodeticient
melanoma
infcxtmn
1” melanoma
IL-8 produced
melanoma
cells was
After
,,roductton
the human
of K-8 on the growth
of the
infiltration.
a potent
that advancedstage
effect
melanoma
of the neutrophd.med~ated
site
of
I” summary,
anti-tumor
OF GENE EXPRESSION
OF PERIPHERAL
BLOGD
Kobe,
Japan Interleolond
ANALYSIS
CELL C!YTOKlNES IN ATOPIC DERMATITIS PATIENTS BY MEANS OF RT-PCR WITH INTERNAL STANDARDS
A. Fujioka, K. Banba, H. Takasu, A. lshibashi, Dept. of Dermatology, National Defense Medical Collcee. T. Toda. Dwf. of Molecular Bioloev. Tokvo Metrocolitan
Pbdadelphia,
School of Mechcine,
QUANTlTATIVE MONONUCLEAR
on
tolls may
of Medicine I” the srudy of mRNA levels of peripheral blood mononuclear cell cytokins in atopic demmtitis o&n&. the conventional RT-PCR method detects the messaees of both ?hl and Th2 cytokines only qualitatively in most cases, without elucidating the immu”“logicai predominance of one over ffie other. In our present study to establish a simple and reliable quant~talive measttrement method, we prepared chimera DNA
.
standards for quantitating IFN- 7 from Thl sod IL-4 from ‘IbZ by amplifying pBR322 DNA using joint primers of human cytokines and the plasmid. The chimera DNAs served as internal standards for PCR. With the mRNA of peripheral blood mo”onuclear cell obtained from atopic detmatifis patients and the healthy control as the mooid, cDNA was synthesized and sobjeued to PCR togethn with the corresponding internal standard, each cytokine placed in one tube. After agamse gel electmphoresis and &idiom bromide staining, the photographic negative picture of tbe specimen was examined by means of densitomcby so as to quantitatively de&nine the expression of cytokins on the basis of their ratio between the internal standard and specimen. As a result, Thl-derived cytokinc was shown to lx more dotizmt in the peripheral blood of patients with chronic atopic dermatitis.
~ytotoxwty.
1088
1091
INCREASED EXPRESSION OF IFN-y IN SMW BOWELBlOPSIES OF PATIENTS WITH SyMpToMAnCGlJJ,‘ENSENSITIVE ENTEROPATHWSE) COMPARED TO PATLENTS
INTERLEUKIN-18 m-18) AND INTERLEUKIN-12 (llx12) SYNERGISTICALLY AUGMENT CELGMEDJA’IED IMMUNITY IN CUTANEOUS T-CELL LYMPHOMA (CZL). EK I&&&i&& Deparnnent of Dermatology, University of Pennsylvania, Philadalnhia PA lmmt&&&&Iities associated with advanced forms of CTCL are chamcteti~d bv marked deficiencies in cell-mediated immwitv and T-heloer tvoe 1 fRt1,
With DERMAnnS HEPzETJFOftMStTXQ. B Bsleti. AD W&_&D Smilien. W Hall. Dt,,h.w,, “A and Duke Medica, Center, Our@ NC “SA. DH patienti have an associated GSE, most oRen wti no ganrcdntcstinaUG1) symptoms, even while on a 8luten contaming diu In mntwt, patients with imiated GSE dwelop Gi symptoms when on a gluten comaining diet hut do twt develop skin disease. We bypothsized daat the diBemt cDnical presematie”s of there loom of GSEarea rewlt of thepatternof cyiekincexpraian in the smallbowel that ecctmwiththe ingestionof@ten Smallbowelbiopsies were obcained from 8 DH patients. alI on gluten contaming diets. 5 padeats with isolated GSE an &ten with xtivs GI symptoms and 4 pauents with is&ted GSE on a @en free diit(GFD) wilhan Gi symptoms. Biopsies were tau, anb mP.NA is&ted. C-DNA was arewed mitt8 reverse trantiowe and PCR tinned with P”. FCR was
&tokirtc production, including interferon gammav(IFN-7). ri-lS:ke k-121 induces IFi+-t production and enhances cytotoxic lymphocyte activity. Since we have show” that IL-12 revetxs the immune defects obsewed in CICL* we investigated the potency of IL-I 8 to synergistically augment IL12 in restoring immune functions in CTCL. Peripheral blood mononocletx cells (PBBMC) from patients with advanced C%!L (n=4) and normal healthy volunteers (“~2) were cultured with medium alone,
IL-18(lOnghnl) or IL-lZ(lng/ml) alone or in combination for 24 hours.
or -12.01 T&a was r&d between pients with DH. symptomatic GSE or asymptomatic GSE Analysis of the IFN-y expression havever reveakd that paoenU with DH an a 8iuten diet had signiticantly lower ratio of IFN+D38 wrpre&n(O.71) than active GSE patienW2 13)( W.05) but r)ot different from asymptomatic patients titb GSE(l.Z2)(pXIO5). Analysis of TGF-B exprcssiott revealed tha active GSE patietientralso had a higher ratio of TGF+/CD3s(4. I) than DH patientsienll(l.22) or asymptamatc GSE patie,,u(O. 18)(p
those patients
in patient PBMC. IL18 alone, however appeared to depress IFN-yproduction in normal PBMC from 658 to 4lOpdt”l and in patient PBMC from 47 to 38pg/ml. When IL-18 was combined with lL-12 a synergistic increase in IFN-yproduction was observed in both normal (1808 to 2338pg/ml) and patient (124 to 165pg/ml) PBMC. Similarly, cultures combining Il.18 and IL12 detnonsuated enhanced NK cell activity in comparison to IL-18 orlL-12 alone. SincelLand IL12 appearto have the capacity to correct some of the immune abnonnalitics associated with advanced Cl’U, our preliminary data have important rhempeutic implications and soppon the rationale fo combine both biologic agents io clinical protocols.
1092
1089 Macrophage Colony Stimulating Factor (M-CSF) human keratinocytes. Martin I.. Johnso~k Cbristoti~ Depts. of DematoI&y
PHA(lfiglml) was then added for the final 24 hours and supernatants wereassayed by ELISA for IFN-7. PBMC were aIs assayed for NK cell activity using cbmmitm51 labeled KS62 cells as targets. When compared to media. IL-12 alone augmented lFN-7 production from 658 to 1808pglml in normal PBMC and from 47 to 124pg/ml
protein production by muine but not Walsh. Katherine A. Kelly, and Immunology, Univ. of Colorado
HSC, Denver. CO, USA; USAFIT, Wright-Patterson AFB. OH, USA; Dept. of Pathology and Immunology, Monash Medical School, Prahmn, Australia. Keratinoeytes actively participate 1” the inklammatory process primarily through the production and secretion of cytokines. One cytokme that has bee” attributed to be part of the cytokine repertoire of mwine and human keratinocytes is macrophage colony stimulating factor or M-CSF. However, previously published data concluding that MCSF is produced by human kerarinocytes is in doubt. By “s~“g Golgi immunostaining, we assessed the expression of M-CSF protein in human primary keratinccytes and the human keratinocyte lines, HaCaT and A431, relative to that in M-CSF-producing celI controls, the nwine keratinocyte lines, PAM212 and XB-2, and the human M-CSFgene transfected murine melanoma Line,B16FlOA2. Intracellular M-CSF protein was identified on an individual cell basis using M-CSF-specific monoclonal antlbodles in pemxablized cells which showed enhanced expression after the addition of monensin for the last 2 hours of culture. M-CSF-producing cells were enumerated by fluorescent microscopy and by stngle-color cytofluommetry. Essentially all of the phorbol acetatestimulated PAM212 control cells and all of the human M-CSF-transfected A2 control cells were M-CSF producers. Smaller numbers of XB-2 cells, control celis that are weak producers of M-CSF, could be. identified as M-CSF producers when these cells were stimulated with phorbol acetate followed by monensin treatment. Enumeration of M-CSF+ cells by flow cytometry, although less sensitive than manual enumerauon of fluorescent cells, and M-CSF bioassays mirrored these findings. However, in contrast to the data of the three different control ceils, M-CSF protein could not be identified in any of the prtmary human keratinocytes “or in the human HaCaT and A43 1 keratinocy~c lines eve” after phorbol acetate stimulation and monensin treatmeot.
~OPHEWTWE CHARA-TloN Gf curANEOIJs lNKrrRATE OF LEsloNAL SKIN BEFORE AND AFTER FXR.4cORFuRF.U PHoIucHEM0-Y IN CTCL PATIENls M. Fimkmi, P. Rubegni, C Miracco’, G. De Aloe, Lm Department of Dermatology, and’ Department of Pathology, University of Siena, Italy. Poticlinim “Lx Scott& 53 100 Siena - ITALY Extracorporeal photochemotherapy release of macrophagedependent currently considered to modulate evaluated the fimction of peripheral
(ECP) has bee” show” to caose proinflammatory cytoldnes and is immune response. I” a previous blood mononuclear cells (PBMC) in
a marked therefore study we 8 patients
with early stage (Ib) c”tnneoos T-4
iymphoma (CTCL). before and one year after ECP. Before treatment. we observed that PBMC aroduced sitmiticantk higher levels of IL4 and lower levels of IF&y t&n in h&thy control subjects.
Afla one year of ECP lI.4 and IFN-y production no longer differed from that of control subjects. We co”clttded that ECP reverses the ThVIh2 imbalance. Here we repoti a retrospective inmunobistochemical study of lesional ski” hefore and after ECP in the same CTCL patients. Immunobistocheutistty wu performed on fured sections using antibodies against CDl% CD3. CD4, CDS. CDS. Mibl, CD30,
CD31, CD45RO and CD45RA by the avidin-biotin complex method. The results showed that ECP is capable 10 modifies the itnmtmopheootype pattern of lesional ski” in6ltrate in early stage CTCL p&nts, pretioosty observed in peripheral blood.
matching
the modifications
that we