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In vitro detection of contact allergens; primary in vitro sensitization
ESDR I JSID I SID Abstracts
0997
1000
IN VITRO DETECTION OF CONTACT ALLERGENS; PRIMARY IN VITRO SENSITIZATION. RS Kalish, JA Wood, Dept, Derm SUNY Q Stony Brook, Stony Brook, NY, USA There is a great need for in vitro methods for testing Primary in vitro sensitization allergenicity of compounds. is the sensitization of naive T-lymphocytes to antigens which This generally requires the donor has ne"er encountered. dendritic antigen presenting cells (i.e. Lanqerhans cells). We have developed a system for primary in vitro sensitization of human T-cells without the use of dendritic AX. Mitomycin C treated Epstein Barr virus (EBV) transformed human B-cells were added a8 a source of co-stimulatory molecules (trans. costimulation). This B-cell line lacks EL?+-DR. and HLA-A,B,C permitting the use of these B-cells with lymphocytes from unrelated donors. Culture of co-stimulatory B-cells with human lymphocytes, monocytes, and allergen induces primary a vitra sensitization of T-lymphocytes to the allergen, which is detectable as 'H-thynidine uptake after 7 days of culture. Peripheral blood monocytes function ae antigen presenting cells in this system, since the co-stimulatory B-cells lack Proliferation was induced to antigen presenting molecules. the contact allergens paraphenylenediamine, balsam of Peru, No response wa8 detected to the and cinnamaldehyde. irritants SDS, Tween 20, or the weak allergen vanillin. This system was able to differentiate moderate allergens from and can and weak from moderate allergens, irritants,
ALTERED GROWTH OF MAST CELLS IN THE ATOPIC ECZEMA-MODEL MOUSE, NC. Takaaki Hiraaun. Eishin Morita. Sa&e Kaneko. Yoshikm Kamevoshi Toshihiko Tanaka md Shoso Yamoto, Department of Dermatology, Hiroshima University School of Medicine, Hiroshima, Japan An inbred strain of mice, NC develop atopic eczema-like skin lesions associated with severe scratch by an infection of the fur mite Myocoptes musculinus. Elevated levels of serum IgE and dermal mast cell hyperplasia are also observed in the fur mite-infested NC. To determine a mechanism for the manifestation of the skin lesions found in NC, we prepared bone marrow-derived mast cells (BMMC) from NC, C57BU6 and BALE/c and compared the properties of these cultured mast cells. Histamine content of BMMC from NC was 0.67 pg/cell and significantly higher than those of C57BU6 (0.09 pg/cell) and BALE/c (0.04 pg/cell), although percent histamine releases in BMMC were found to be similar in these three strains. Adhesion of BMMC to plastic dishes was higher in NC compared with C57BU6 and BALE/c. BMMC obtained from NC were sustained for more than 4 weeks in the presence of 100 nglml stem cell factor, whereas those from C57W6 and BALE/c died within 2 weeks. These results suggest that the altered growth of mast cells partially accounts for an increase of dermal mast cells and hypersensitivity to fur mites found in NC.
potentially
act
as an in vitro
screen
for
allergenicity.
0998
1001
MOUSE 3T3 FIBROBLASTS INDUCE MOUSE BONE MARROW-DERIVED MASTCELLS TO AQUIRE RESPONSNENESS TO SUBSTANCE P Eishin i Shoso Motita. Takaaki Hiraaun. ToshihikoTanaka. Yamamoto, Department of Dermatology, Hiroshima University School of Medicine, Hiroshima, Japan. Connective tissue-type mast cslls (CTMC), that reside in serosal cavities and skin, dagranulate in response to neurogenic peptides, such as substance P. However, mouse mast cells obtained by culturing hematopoietic cells in vitro with IL-3 and IL-4 (BMMC) lack responsiveness to substance P. EMMC co-cultured with 3T3 fibroblasts differentiate morphologically to become CTMC-lilte, indicating that the growth of mast cells are strongly influenced by the connective tissue microenvironment. To determine if 3T3 fibroblasts can influence the responsiveness to neumgenic peptides, BMMC were cocultured with a confluent monolayer of NIH3T3 fibroblastsand assayed for substance P-induced histamine release. After 3 weeks in coculture in the presence of stem cell factor, BMMC showed positive staining with safranin. and released histamine in response to substance P (1O9to lo4 M). This was not seen in culture with stem cell factor alone. These results show Mat coculture with 3T3 fibroblasts cause a differentiation of BMMC into CTMC, in function, as well as in morphology.
PHENOTYPIC
ANALYSIS OF HUMAN LANGERHANS
CELLS AFTER
The need to develop in vitro predictive tests that could &n&y potential sllergens has been recognized for many years. Langerhans cells (LC) play a major role in the development of hypersensitivity contact reactions by picking up the hapten within epidermis and migrating to regional lymph node where they trigger specific T cell activation. llte aim of the present study was to analyze whether contact of human L.C with a strong allergen could induce specific phenotypic changes, ss compared to those observed with an irritant Human epidennal cells were obtained through Vypsination (trypsin 0.05%. 1 h, 37’C!)of normal skin samples and enriched for LC (eLC) by density centifugation. eLC suspensions, containing 50 to 70% IX, were treated with either trinitmbenzene sulfonic acid (TNP, 5 mM) or sodium lauryl sulfate (SLS, 10 pg/ml) for 10 mn at 37°C. then washed and further incubated for 2 or 18 h at 37’C. Cells were then phenotyped using different monoclonsl antibodies and ansly& on a FACS scan. Results showed that after 18 h. no significant alterations of HLA-DR. ICAM-1, B7-I. B7-2 or E-cadherin expression were noticed whether the cells had been treated with either SLS or TNP. By contmst, and ss previously reported, TNP but not SLS treatment induced a reduced expression of HLA-DR expression on 2 b-incubated Lx: (p
0999
1002
EVIDENCE FOR ANTIGEN RESTRICTION IN PERIPHERAL BLOOD * * LYMPHOCYTES IN PsoRIAsIS. S J&J&. St John’s Institute of Dermatology,and *Tissue Typing Laborstory, UMDST London. Most memory T-lymphocytes in lesional psoriatic skin express surface bound cutaneouslymphocyteassociated antigen (CLA). Restriction of T-cell receptor (TCR) Vp chain usage by tymphcqtes in psoriatic skin has been shown. It is unknown if this represents skin homing or local expansion of VP restricted lymphocytes, or whether it reflects Vp restriction of CLA positive T-cells in peripheral blood. To address this issue peripheral blood lymphocytes (PBL) from healthy vohmteers (n=S) and patients with chronicplapue psoriasis (n=8) were assessed by flow cytometry using monoclonal antibodies to CLA and eight TCR VP families (VPZ, 3.1, 5.1, 5.2/3, 8.1/2, 12.1, 13 l/3, 17). Psoriasis patients demonstrsted increased CLA expression (mean=1138%) compsred to normals (mesn=8.32%) There wss a significant increase in VP2 positive PBL within the CLA positive populationin psoriasispatients(mean 7 68%) compared to normals (mean 6.21%); p
AN IMPROVED METHOD FOR GENERATING LARGE QUANTITIES OF DENDRITIC CELLS FROM MOUSE BONE MARROW AND NOVEL FACTORS REGULATING THEIR MATURATION ~ Lutz. NA Kukutsch. AL.l Oeilvie. S R6!3ner.G Sclu&, Department of Dermatology, University of Erlsngen, Germany Dendritic cells (DC) are scarce leukocytes specialized to sensitize T cells in viva Therefore DC research was promoted by the method to generate DC from bone marrow with GM-CSF, where about 5 x lo6 DC develop per mouse in 1 week. We modified this technique in order to generate much larger numbers (> 100 x 106) of MHC II+, CD1 I& BM-DC per mouse. Our culture method is primarily based on seeding BM precursors at lower density st the onset, but extending the culture periods to 10-14 days. Furthermore we avoid any active depletion of BM cell subpopulations that presumably results in loss of precursors. The majority of non-adherent contaminating cells dies out over the prolonged culture period or by reducing the concentration of GM-CSF, so that BM-DC purity is 90-958 around day 10. Detailed analysis (e.g. MHC class II expression, antigen uptake and processing) of the maturational stages of BM-DC revealed that a continuous balance of immature and mature DC, rather than a progressive maturation develops. Uniform maturation of DC (i.e. increase in stimulatory capacity, downregulation of antigen processing) can, however, be induced by appropriate stimuli like LPS and TNF alpha. In contrast, lower cell density, high LPS levels at the beginning of the BM-DC culture, and very low concentrations of GM-CSF allow the selective generation of immature BM-DC Our modified method thus permits the simple and cost-effective in vitro generation of virtually unlinuted numbers of murine DC at defined stages of maturation, snd ~111prove particularly valuable to study DC at the moleculsr level.
0997
1000
IN VITRO DETECTION OF CONTACT ALLERGENS; PRIMARY IN VITRO SENSITIZATION. RS Kalish, JA Wood, Dept, Derm SUNY Q Stony Brook, Stony Brook, NY, USA There is a great need for in vitro methods for testing Primary in vitro sensitization allergenicity of compounds. is the sensitization of naive T-lymphocytes to antigens which This generally requires the donor has ne"er encountered. dendritic antigen presenting cells (i.e. Lanqerhans cells). We have developed a system for primary in vitro sensitization of human T-cells without the use of dendritic AX. Mitomycin C treated Epstein Barr virus (EBV) transformed human B-cells were added a8 a source of co-stimulatory molecules (trans. costimulation). This B-cell line lacks EL?+-DR. and HLA-A,B,C permitting the use of these B-cells with lymphocytes from unrelated donors. Culture of co-stimulatory B-cells with human lymphocytes, monocytes, and allergen induces primary a vitra sensitization of T-lymphocytes to the allergen, which is detectable as 'H-thynidine uptake after 7 days of culture. Peripheral blood monocytes function ae antigen presenting cells in this system, since the co-stimulatory B-cells lack Proliferation was induced to antigen presenting molecules. the contact allergens paraphenylenediamine, balsam of Peru, No response wa8 detected to the and cinnamaldehyde. irritants SDS, Tween 20, or the weak allergen vanillin. This system was able to differentiate moderate allergens from and can and weak from moderate allergens, irritants,
ALTERED GROWTH OF MAST CELLS IN THE ATOPIC ECZEMA-MODEL MOUSE, NC. Takaaki Hiraaun. Eishin Morita. Sa&e Kaneko. Yoshikm Kamevoshi Toshihiko Tanaka md Shoso Yamoto, Department of Dermatology, Hiroshima University School of Medicine, Hiroshima, Japan An inbred strain of mice, NC develop atopic eczema-like skin lesions associated with severe scratch by an infection of the fur mite Myocoptes musculinus. Elevated levels of serum IgE and dermal mast cell hyperplasia are also observed in the fur mite-infested NC. To determine a mechanism for the manifestation of the skin lesions found in NC, we prepared bone marrow-derived mast cells (BMMC) from NC, C57BU6 and BALE/c and compared the properties of these cultured mast cells. Histamine content of BMMC from NC was 0.67 pg/cell and significantly higher than those of C57BU6 (0.09 pg/cell) and BALE/c (0.04 pg/cell), although percent histamine releases in BMMC were found to be similar in these three strains. Adhesion of BMMC to plastic dishes was higher in NC compared with C57BU6 and BALE/c. BMMC obtained from NC were sustained for more than 4 weeks in the presence of 100 nglml stem cell factor, whereas those from C57W6 and BALE/c died within 2 weeks. These results suggest that the altered growth of mast cells partially accounts for an increase of dermal mast cells and hypersensitivity to fur mites found in NC.
potentially
act
as an in vitro
screen
for
allergenicity.
0998
1001
MOUSE 3T3 FIBROBLASTS INDUCE MOUSE BONE MARROW-DERIVED MASTCELLS TO AQUIRE RESPONSNENESS TO SUBSTANCE P Eishin i Shoso Motita. Takaaki Hiraaun. ToshihikoTanaka. Yamamoto, Department of Dermatology, Hiroshima University School of Medicine, Hiroshima, Japan. Connective tissue-type mast cslls (CTMC), that reside in serosal cavities and skin, dagranulate in response to neurogenic peptides, such as substance P. However, mouse mast cells obtained by culturing hematopoietic cells in vitro with IL-3 and IL-4 (BMMC) lack responsiveness to substance P. EMMC co-cultured with 3T3 fibroblasts differentiate morphologically to become CTMC-lilte, indicating that the growth of mast cells are strongly influenced by the connective tissue microenvironment. To determine if 3T3 fibroblasts can influence the responsiveness to neumgenic peptides, BMMC were cocultured with a confluent monolayer of NIH3T3 fibroblastsand assayed for substance P-induced histamine release. After 3 weeks in coculture in the presence of stem cell factor, BMMC showed positive staining with safranin. and released histamine in response to substance P (1O9to lo4 M). This was not seen in culture with stem cell factor alone. These results show Mat coculture with 3T3 fibroblasts cause a differentiation of BMMC into CTMC, in function, as well as in morphology.
PHENOTYPIC
ANALYSIS OF HUMAN LANGERHANS
CELLS AFTER
The need to develop in vitro predictive tests that could &n&y potential sllergens has been recognized for many years. Langerhans cells (LC) play a major role in the development of hypersensitivity contact reactions by picking up the hapten within epidermis and migrating to regional lymph node where they trigger specific T cell activation. llte aim of the present study was to analyze whether contact of human L.C with a strong allergen could induce specific phenotypic changes, ss compared to those observed with an irritant Human epidennal cells were obtained through Vypsination (trypsin 0.05%. 1 h, 37’C!)of normal skin samples and enriched for LC (eLC) by density centifugation. eLC suspensions, containing 50 to 70% IX, were treated with either trinitmbenzene sulfonic acid (TNP, 5 mM) or sodium lauryl sulfate (SLS, 10 pg/ml) for 10 mn at 37°C. then washed and further incubated for 2 or 18 h at 37’C. Cells were then phenotyped using different monoclonsl antibodies and ansly& on a FACS scan. Results showed that after 18 h. no significant alterations of HLA-DR. ICAM-1, B7-I. B7-2 or E-cadherin expression were noticed whether the cells had been treated with either SLS or TNP. By contmst, and ss previously reported, TNP but not SLS treatment induced a reduced expression of HLA-DR expression on 2 b-incubated Lx: (p
0999
1002
EVIDENCE FOR ANTIGEN RESTRICTION IN PERIPHERAL BLOOD * * LYMPHOCYTES IN PsoRIAsIS. S J&J&. St John’s Institute of Dermatology,and *Tissue Typing Laborstory, UMDST London. Most memory T-lymphocytes in lesional psoriatic skin express surface bound cutaneouslymphocyteassociated antigen (CLA). Restriction of T-cell receptor (TCR) Vp chain usage by tymphcqtes in psoriatic skin has been shown. It is unknown if this represents skin homing or local expansion of VP restricted lymphocytes, or whether it reflects Vp restriction of CLA positive T-cells in peripheral blood. To address this issue peripheral blood lymphocytes (PBL) from healthy vohmteers (n=S) and patients with chronicplapue psoriasis (n=8) were assessed by flow cytometry using monoclonal antibodies to CLA and eight TCR VP families (VPZ, 3.1, 5.1, 5.2/3, 8.1/2, 12.1, 13 l/3, 17). Psoriasis patients demonstrsted increased CLA expression (mean=1138%) compsred to normals (mesn=8.32%) There wss a significant increase in VP2 positive PBL within the CLA positive populationin psoriasispatients(mean 7 68%) compared to normals (mean 6.21%); p
AN IMPROVED METHOD FOR GENERATING LARGE QUANTITIES OF DENDRITIC CELLS FROM MOUSE BONE MARROW AND NOVEL FACTORS REGULATING THEIR MATURATION ~ Lutz. NA Kukutsch. AL.l Oeilvie. S R6!3ner.G Sclu&, Department of Dermatology, University of Erlsngen, Germany Dendritic cells (DC) are scarce leukocytes specialized to sensitize T cells in viva Therefore DC research was promoted by the method to generate DC from bone marrow with GM-CSF, where about 5 x lo6 DC develop per mouse in 1 week. We modified this technique in order to generate much larger numbers (> 100 x 106) of MHC II+, CD1 I& BM-DC per mouse. Our culture method is primarily based on seeding BM precursors at lower density st the onset, but extending the culture periods to 10-14 days. Furthermore we avoid any active depletion of BM cell subpopulations that presumably results in loss of precursors. The majority of non-adherent contaminating cells dies out over the prolonged culture period or by reducing the concentration of GM-CSF, so that BM-DC purity is 90-958 around day 10. Detailed analysis (e.g. MHC class II expression, antigen uptake and processing) of the maturational stages of BM-DC revealed that a continuous balance of immature and mature DC, rather than a progressive maturation develops. Uniform maturation of DC (i.e. increase in stimulatory capacity, downregulation of antigen processing) can, however, be induced by appropriate stimuli like LPS and TNF alpha. In contrast, lower cell density, high LPS levels at the beginning of the BM-DC culture, and very low concentrations of GM-CSF allow the selective generation of immature BM-DC Our modified method thus permits the simple and cost-effective in vitro generation of virtually unlinuted numbers of murine DC at defined stages of maturation, snd ~111prove particularly valuable to study DC at the moleculsr level.