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Cellular approach for detecting narcolepsy-specific alterations in DR2 haplocytes
Cellular Approach for Detecting Narcolepsyspecific Alterations in DR2 Haplotypes P. Strohma/er, G. Mueller-Eckhardr, and K. Meier-Ewert
ABSTRAC-~': In an attempt to detect diseaJe-associated genetic rariations and/or exogenously induced alterations in DR2 haplotypes of narcolepsy patients, primed lymphocytes (PLs) were generated in nine families against DRY. haplotypes of narcolepsy patients and healthy family members. Twenty-four PL reagents were obtained and restimulated by calls of unrelated narcolepsy patients and DR21Dw2-positive healthy individuals. The mean restimulation rates triggered by cells of patients or healtisy controls, respectively, never differed significantly. In primary AILC as w d l as P L T combinations of patients and their HI, A-identical siblings no significant stimulation was obeervj in both directions. We conclude that narcolepsy-specific alterations , f class 11 molecules cannot be cellularly detected or do not exist on peripheral lymphocytes. ABBREVIATIONS CM Culture medium CPM Count per minute MLC Mixedlymphocyte culture PL Primedlymphocyte PLT Primedlymphocyte typing
RC Responder cell RFLP Restriction fragment length polymorphism SC Stimulatorcell
INTRODUCTION Recent findings of a nearly 100% association between narcolepsy and HLA-DR2/ Dw2 [1-10] have attracted considerable attention. More than any other disease, narcolepsy appears suitable as a model for studying the possible mechanism(s) of HLA and disease associations. Furthermore, new information on the pathogenetic background of a disease of as-yet unknown origin can be anticipated. We have recendy observed that in narcoleptic males, but not in females, the transmission of DR2 haplotypes deviates significandy from Mendelian expectation [11]. This finding prompted us to search for a disease-associated genetic variation of the DR2 molecule, or ofgene products of closely linked loci, which would explain the observed phenomenon. On the other hand, our experience that muldcase narcolepsy families seem to be rather rare has led us to the question whether exogenous factors might be able From the Institute for Clinical Immunology and Transfusion Medicine, Juaus Liebig University, D6300 Giessen (P.S.; G.M.-E.) and the Neurological Hospital and Sleep Disorder Unit, D-3578 Sc#waltratadt, Federal Republic of Germany (K.M.-E.). Address reprint requests to: Genrud Muefler-Eckhardt, M.D.. Institute for Clinical Immunology and Transfusion Medidne, Justus Lt'ebig University, Langhansstrasae 7, 1)-6300 Giessen, FederalRepublic of Germany. ReceivedDecember21, 1987; revisedApril 12, 1988.
HumanImmunology2Z, 221-225(1988) ©AmericanSocietyfor Histccompatibilityand Immunogenedcs,I988
22 l 0198-8859/88/53,50
222
p. Strohmaier, G. Mueller-Eckhardt, and K. Meier-Ewert to alterate specifically DR2 molecules or haplotypes, respectively, resulting in the generation of an "altered self" and potential induction of autoimmunity. We have, therefore, chosen a cellular approach to detect narcolepsy specific alterations of class II molecules. Lymphocytes were primed against narcolepsyassociated DR2 haplotypes in families and restimulated alternatively by cells of unrelated narcolepsy patients or healthy DR2/Dw2-positive individuals. In addition, mixed lymphocyte cultures (MLCs) and priming experiments were carried out between patients and their genotypically identical healthy siblings.
MATERIALS AND METHODS From a total of 67 narcolepsy patients and 18 families investigated recently [5,9,11], 9 informative families as well as 26 unrelated patients were selected for the study. One family and 14 unrelated healthy DR2/Dw2-positive individuals served as controls. Priming combinations were selected such that both stimulator cells (SCs) and responder ceUs (RCs) shared one genotypically identical haplotype, but were different for the DR2 haplotype (Figure 1). A total of 106 irradiated (20 gy) SCs were cocultured with 10~RCs in 2 ral culture medium in 24-well culture plates (Costar) for 10-11 days at 37°C with 5% COy Thereafter cells were either frozen or restimulated with 3 x 106 irradiated (40 gy) cells of the original SC donor for 5 days. Nineteen PL reagents against DR2/Dw2 haplotypes of narcolepsy patients and five PLs against those of healthy individuals were tested in second-degree MLCs (PLT) [12] against a total of 26 unrelated narcolepsy patients and 14 normal controls. A total of 2 x 105 PLs were cocultured in triplicates with 106 SCs in 1003/~1 culture medium (CM) for 72 hr at 37°C with 5% CO2. One microcurie of H-thymidine (5 Ci/mmol, Amersham) per well was added 6 hr before cells were harvested. 3H-thymidine incorporation was measured in counts per minute (Cpm) and calculated as mean of triplicates. For statistical comparison the mean restimulation rates of DR2/Dw2-posidve narcolepsy patients or controls, respectively, were calculated. The null hypothesis of the equality of the two means was .,~amined by the t-test modified by Welch [14]. Mixed lymphocyte cultures [ 13] were performed with ten different combinations out of three families (numbers 01, 03, 17 in Figure 1) between HLA A,B,C,DR,DQ-identicai siblings, one of whom was a narcolepsy patient and the other of whom was healthy. Cultures were set up for 5, 6 and 7 days in U form 96-well microtiter plates (Greiner) using 105 irradiated (20 gy) SCs and 105 RCs in 100/zl CM. RESULTS A N D DISCUSSION PL-generated against narcolepsy associated or healthy DR2 haplotypes in families (Figure 1) did nor show signficant differences when restimulated by cells of narcolepsy patients or DR2/Dw2-positive controls. The mean restimuladon rate triggered by patients' cells was slightly higher in 14 of 19 experiments with PL against narcolepsy-associated haplotypes, but also in three out of five experiments with reagents produced against DR2 haplotypes of healthy family members. Thus we did not observe a cellular reaction pattern that would quantitatively or qualitatively discriminate narcolepsy-associated haplotypes from those of healthy DR2/Dw2-positive controls. MLC as well as PLT experiments between HLA-A,B,C,DR,DQ identical siblings in three families did not reveal detectable stimulation or a priming effect in either direction.
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FIGURE 1 Pedigrees of families from which priming and MLC combinations were chosen. Asterisk, DR2 haplotypes; hatched symbols, narcolepsy patients; open symbols, healthy family members. Numbers indicate individuals included in ff,e study. To search for narcolepsy-associated genetic variations of DR2 hap|otypes, several groups have investigated the DNA polymorphism of the DR/DQ region in patients and controls [15-18]. In addition to the fact that narcolepsy patients of all populations showed exclusively the RFLP pattern specific for the cellular determinant Dw2, no additional variation occurring in patients but not in healthy controls was discernible. Nevertheless, investigations at the cellular level, namely of the class II molecule(s) potentially involved, a~ght provide additional information. Furthermore, exogenous alterations of HLA molecules would more l/kely be recognized by such means. Mareusson and Mtller, using a similar approach as ours, have produced PLT reagents against DR7/Dw7 haplotypes in psoriasis families [19]. With some reagents, these authors noticed higher and more frequent responses by restim-
224
P. Strohmaier, G, Mueller-Eckhardt, and K. Meier-Ewert ulation with DRT-positive cells of psoriasis patients as compared to DRT-positive controls. This was interpreted to mean that these reagents recognized at least partly different DRT-associated determinants in patients versus controls. However, it cannot be excluded that these differences reflect solely different DR7-relared cellular determinants such as DwT, Dw11, Dw17, in the control group. Considering only Dw2-positive, Dwl2-negative control cells for statistical evaluation we were unable to verify a significant cellular discrimination of narcolepsy-associated DR2 haplotypes from those of healthy individuals. We conclude from our study that narcolepsy-specific genetic variations and/or exogenous alterations of DR?. molecules (or of gene produe~s of closely linked loci) cannot be cellularly detected on peripheral lymphocytes. However, this does not exclude exogenously caused alterations, which could be located at a certain (yet unknown) site of the central nervous system and induce local immune reactions in vivo. Because even the exact local site of pathological events within the central nervous system leading to clinical signs of narcolepsy is still unknown, the pathogenetic background of the clos,.~ association between HLA and narcolepsy remains speculative.
ACKNOWLEDGMENTS The secretarial expertise of Ms. T. Ille is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft(Mu 725/1-2).
REFERENCES 1. Juji T, Satake M, Honda Y, Doi Y: HLA antigens in Japanese patients with narcolepsy. All the patients were DR2 positive. Tissue Antigens 24:316, 1984. 2. Langdon N, Welsh KI, van Dam M, Vaughan RW, Parkes D: Genetic markers in narkolepsy. Lancet ii: 1178, 1984. 3. Billiard M, SeignaletJ: Extraordinaryassociationbetween HLA-DR2 and narcolepsy. Lancet i: 226, 1985. 4. Matsuki K, Juji T, Tokunaga K, Naohara T, Satake M, Honda Y: Human Histocompatability Leukocyte Antigen (HLA) haplotype frequencies estimated from the data on HLA class I, lI, and Iii antigens in 111Japanese narcoleptics.J Clin Invest 76:2078, 1985. 5. Mueller-Eckhardt G, Meier-Ewert K, Schendel DJ, Reinecker FB, Multhoff G, Mueller-Eckhardt C: HLA and narcolepsy in a German population. Tissue Antigens 28:163, 1986. 6. Poirier G, MonplaisirJ, D6cary F, Mom~geD, Lebrun A: HLA antigensin narcolepsy and idiopathic central nervous system hypersomnolence.Sleep 9:153, 1986. 7. Kramer RE, Dinner DS, Braun WE, Zachary AA, Teresi GA: HLA-DR2 and narcolepsy. Arch Neuro144:853, 1987. 8. Confavreux C, Gebuhrer L, Betuel H, Freidel C, Basmji H, Aimard G, Devic M, Jouvet M: HLA-DR2 negative narcolepsy.J Neurol Neurnsurg Psychiatry 50:635, 1987. 9. Meier-EwertK, Mueller-Eckhardt G, Schendel DJ: Narcolepsy and HLA in the Federal Republic of Germany: populationand familydata. In Honda Y, Juji T (eds): HLA in Narcolepsy. New York, Springer.
Narcolepsy-specific Alterations in DR2 Haplotypes?
225
I0. Wilner A, Steinman L, Lavie P, Peled R, [:riedmann A, Brautbar C: Narcolepsy-cataplexy in IsraeliJews is associated exclusivelywith the HLA DR2 haplotype. A study at the serological and genomic level. Hum lmmunol 21:15, 1988. 11. Mueller-Eckhardt G, Strohmaier P, Schendel DJ, Meier-Ewert K, Mueller-Eckhardt C: Possible male segregation distortion of DR2 haplowpes in narcohpsy patients. Hum Immunol 20:189, 1987. 12. Sheehy M, Soadel P, Mach M, Wank R, Bach F: HLA typing: A rapid assay using primed lymphocytes. Science 188:1308, 1975. 13. Bach FH, Hirschhorn K: Lymphocyte interaction. A potential histocompatabilitytest in vitro. Science 143:813, 1964. 14. Sachs L: Applied Statistics. New York, Springer, 1982. 15. Marcadet A, Gebuhrer L, Betuel H, Seignalet J, Freidel AC, Confavremt C, Billiard M, Daosset J, Cohen D: DNA polymorphism related to HLA-DR2 Dw2 in padents with narcolepsy, lmmunogenetics 22:679, 1985. 16. lnoko H, Ando A, Tsuji K, Matsuki K, Juji T, Honda Y: HLA=D, chain DNA restriction fragments can differentiate between healthy and narcoleptic individuals with HLA-DR2. Immunogenetics 23:126, 1986. 17. Andreas-Zietz A, Keller E, Scholz S, Albert ED, Roth B, Nevsimalova S, Sonka K, Docekal P, lvaskova E, Sdmiz H, Geisler P: DR2-negarive narcolepsy. Lancet i: 684, 1986. 18. Matsuki K. Maeda l-t, Juji T, Inoko H, Ando A, Tsuji K, Honda Y: Taq 1-generated HLA-DQ polymorphism in Japanese padents with narcolepsy, ln~raunogenedcs 27:87, 1988. 19. Marcusson JA, MOiler E: Further studies on psoriasis-associated HLA-Dw/DR.7 using PIT cells primed against a familial psoriasis-linked HLA-haplotype. Tissue Antigens 27:1, 1986.
ABSTRAC-~': In an attempt to detect diseaJe-associated genetic rariations and/or exogenously induced alterations in DR2 haplotypes of narcolepsy patients, primed lymphocytes (PLs) were generated in nine families against DRY. haplotypes of narcolepsy patients and healthy family members. Twenty-four PL reagents were obtained and restimulated by calls of unrelated narcolepsy patients and DR21Dw2-positive healthy individuals. The mean restimulation rates triggered by cells of patients or healtisy controls, respectively, never differed significantly. In primary AILC as w d l as P L T combinations of patients and their HI, A-identical siblings no significant stimulation was obeervj in both directions. We conclude that narcolepsy-specific alterations , f class 11 molecules cannot be cellularly detected or do not exist on peripheral lymphocytes. ABBREVIATIONS CM Culture medium CPM Count per minute MLC Mixedlymphocyte culture PL Primedlymphocyte PLT Primedlymphocyte typing
RC Responder cell RFLP Restriction fragment length polymorphism SC Stimulatorcell
INTRODUCTION Recent findings of a nearly 100% association between narcolepsy and HLA-DR2/ Dw2 [1-10] have attracted considerable attention. More than any other disease, narcolepsy appears suitable as a model for studying the possible mechanism(s) of HLA and disease associations. Furthermore, new information on the pathogenetic background of a disease of as-yet unknown origin can be anticipated. We have recendy observed that in narcoleptic males, but not in females, the transmission of DR2 haplotypes deviates significandy from Mendelian expectation [11]. This finding prompted us to search for a disease-associated genetic variation of the DR2 molecule, or ofgene products of closely linked loci, which would explain the observed phenomenon. On the other hand, our experience that muldcase narcolepsy families seem to be rather rare has led us to the question whether exogenous factors might be able From the Institute for Clinical Immunology and Transfusion Medicine, Juaus Liebig University, D6300 Giessen (P.S.; G.M.-E.) and the Neurological Hospital and Sleep Disorder Unit, D-3578 Sc#waltratadt, Federal Republic of Germany (K.M.-E.). Address reprint requests to: Genrud Muefler-Eckhardt, M.D.. Institute for Clinical Immunology and Transfusion Medidne, Justus Lt'ebig University, Langhansstrasae 7, 1)-6300 Giessen, FederalRepublic of Germany. ReceivedDecember21, 1987; revisedApril 12, 1988.
HumanImmunology2Z, 221-225(1988) ©AmericanSocietyfor Histccompatibilityand Immunogenedcs,I988
22 l 0198-8859/88/53,50
222
p. Strohmaier, G. Mueller-Eckhardt, and K. Meier-Ewert to alterate specifically DR2 molecules or haplotypes, respectively, resulting in the generation of an "altered self" and potential induction of autoimmunity. We have, therefore, chosen a cellular approach to detect narcolepsy specific alterations of class II molecules. Lymphocytes were primed against narcolepsyassociated DR2 haplotypes in families and restimulated alternatively by cells of unrelated narcolepsy patients or healthy DR2/Dw2-positive individuals. In addition, mixed lymphocyte cultures (MLCs) and priming experiments were carried out between patients and their genotypically identical healthy siblings.
MATERIALS AND METHODS From a total of 67 narcolepsy patients and 18 families investigated recently [5,9,11], 9 informative families as well as 26 unrelated patients were selected for the study. One family and 14 unrelated healthy DR2/Dw2-positive individuals served as controls. Priming combinations were selected such that both stimulator cells (SCs) and responder ceUs (RCs) shared one genotypically identical haplotype, but were different for the DR2 haplotype (Figure 1). A total of 106 irradiated (20 gy) SCs were cocultured with 10~RCs in 2 ral culture medium in 24-well culture plates (Costar) for 10-11 days at 37°C with 5% COy Thereafter cells were either frozen or restimulated with 3 x 106 irradiated (40 gy) cells of the original SC donor for 5 days. Nineteen PL reagents against DR2/Dw2 haplotypes of narcolepsy patients and five PLs against those of healthy individuals were tested in second-degree MLCs (PLT) [12] against a total of 26 unrelated narcolepsy patients and 14 normal controls. A total of 2 x 105 PLs were cocultured in triplicates with 106 SCs in 1003/~1 culture medium (CM) for 72 hr at 37°C with 5% CO2. One microcurie of H-thymidine (5 Ci/mmol, Amersham) per well was added 6 hr before cells were harvested. 3H-thymidine incorporation was measured in counts per minute (Cpm) and calculated as mean of triplicates. For statistical comparison the mean restimulation rates of DR2/Dw2-posidve narcolepsy patients or controls, respectively, were calculated. The null hypothesis of the equality of the two means was .,~amined by the t-test modified by Welch [14]. Mixed lymphocyte cultures [ 13] were performed with ten different combinations out of three families (numbers 01, 03, 17 in Figure 1) between HLA A,B,C,DR,DQ-identicai siblings, one of whom was a narcolepsy patient and the other of whom was healthy. Cultures were set up for 5, 6 and 7 days in U form 96-well microtiter plates (Greiner) using 105 irradiated (20 gy) SCs and 105 RCs in 100/zl CM. RESULTS A N D DISCUSSION PL-generated against narcolepsy associated or healthy DR2 haplotypes in families (Figure 1) did nor show signficant differences when restimulated by cells of narcolepsy patients or DR2/Dw2-positive controls. The mean restimuladon rate triggered by patients' cells was slightly higher in 14 of 19 experiments with PL against narcolepsy-associated haplotypes, but also in three out of five experiments with reagents produced against DR2 haplotypes of healthy family members. Thus we did not observe a cellular reaction pattern that would quantitatively or qualitatively discriminate narcolepsy-associated haplotypes from those of healthy DR2/Dw2-positive controls. MLC as well as PLT experiments between HLA-A,B,C,DR,DQ identical siblings in three families did not reveal detectable stimulation or a priming effect in either direction.
Narcolepsy-specificAlterations in DR2 Haplo~pes?
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FIGURE 1 Pedigrees of families from which priming and MLC combinations were chosen. Asterisk, DR2 haplotypes; hatched symbols, narcolepsy patients; open symbols, healthy family members. Numbers indicate individuals included in ff,e study. To search for narcolepsy-associated genetic variations of DR2 hap|otypes, several groups have investigated the DNA polymorphism of the DR/DQ region in patients and controls [15-18]. In addition to the fact that narcolepsy patients of all populations showed exclusively the RFLP pattern specific for the cellular determinant Dw2, no additional variation occurring in patients but not in healthy controls was discernible. Nevertheless, investigations at the cellular level, namely of the class II molecule(s) potentially involved, a~ght provide additional information. Furthermore, exogenous alterations of HLA molecules would more l/kely be recognized by such means. Mareusson and Mtller, using a similar approach as ours, have produced PLT reagents against DR7/Dw7 haplotypes in psoriasis families [19]. With some reagents, these authors noticed higher and more frequent responses by restim-
224
P. Strohmaier, G, Mueller-Eckhardt, and K. Meier-Ewert ulation with DRT-positive cells of psoriasis patients as compared to DRT-positive controls. This was interpreted to mean that these reagents recognized at least partly different DRT-associated determinants in patients versus controls. However, it cannot be excluded that these differences reflect solely different DR7-relared cellular determinants such as DwT, Dw11, Dw17, in the control group. Considering only Dw2-positive, Dwl2-negative control cells for statistical evaluation we were unable to verify a significant cellular discrimination of narcolepsy-associated DR2 haplotypes from those of healthy individuals. We conclude from our study that narcolepsy-specific genetic variations and/or exogenous alterations of DR?. molecules (or of gene produe~s of closely linked loci) cannot be cellularly detected on peripheral lymphocytes. However, this does not exclude exogenously caused alterations, which could be located at a certain (yet unknown) site of the central nervous system and induce local immune reactions in vivo. Because even the exact local site of pathological events within the central nervous system leading to clinical signs of narcolepsy is still unknown, the pathogenetic background of the clos,.~ association between HLA and narcolepsy remains speculative.
ACKNOWLEDGMENTS The secretarial expertise of Ms. T. Ille is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft(Mu 725/1-2).
REFERENCES 1. Juji T, Satake M, Honda Y, Doi Y: HLA antigens in Japanese patients with narcolepsy. All the patients were DR2 positive. Tissue Antigens 24:316, 1984. 2. Langdon N, Welsh KI, van Dam M, Vaughan RW, Parkes D: Genetic markers in narkolepsy. Lancet ii: 1178, 1984. 3. Billiard M, SeignaletJ: Extraordinaryassociationbetween HLA-DR2 and narcolepsy. Lancet i: 226, 1985. 4. Matsuki K, Juji T, Tokunaga K, Naohara T, Satake M, Honda Y: Human Histocompatability Leukocyte Antigen (HLA) haplotype frequencies estimated from the data on HLA class I, lI, and Iii antigens in 111Japanese narcoleptics.J Clin Invest 76:2078, 1985. 5. Mueller-Eckhardt G, Meier-Ewert K, Schendel DJ, Reinecker FB, Multhoff G, Mueller-Eckhardt C: HLA and narcolepsy in a German population. Tissue Antigens 28:163, 1986. 6. Poirier G, MonplaisirJ, D6cary F, Mom~geD, Lebrun A: HLA antigensin narcolepsy and idiopathic central nervous system hypersomnolence.Sleep 9:153, 1986. 7. Kramer RE, Dinner DS, Braun WE, Zachary AA, Teresi GA: HLA-DR2 and narcolepsy. Arch Neuro144:853, 1987. 8. Confavreux C, Gebuhrer L, Betuel H, Freidel C, Basmji H, Aimard G, Devic M, Jouvet M: HLA-DR2 negative narcolepsy.J Neurol Neurnsurg Psychiatry 50:635, 1987. 9. Meier-EwertK, Mueller-Eckhardt G, Schendel DJ: Narcolepsy and HLA in the Federal Republic of Germany: populationand familydata. In Honda Y, Juji T (eds): HLA in Narcolepsy. New York, Springer.
Narcolepsy-specific Alterations in DR2 Haplotypes?
225
I0. Wilner A, Steinman L, Lavie P, Peled R, [:riedmann A, Brautbar C: Narcolepsy-cataplexy in IsraeliJews is associated exclusivelywith the HLA DR2 haplotype. A study at the serological and genomic level. Hum lmmunol 21:15, 1988. 11. Mueller-Eckhardt G, Strohmaier P, Schendel DJ, Meier-Ewert K, Mueller-Eckhardt C: Possible male segregation distortion of DR2 haplowpes in narcohpsy patients. Hum Immunol 20:189, 1987. 12. Sheehy M, Soadel P, Mach M, Wank R, Bach F: HLA typing: A rapid assay using primed lymphocytes. Science 188:1308, 1975. 13. Bach FH, Hirschhorn K: Lymphocyte interaction. A potential histocompatabilitytest in vitro. Science 143:813, 1964. 14. Sachs L: Applied Statistics. New York, Springer, 1982. 15. Marcadet A, Gebuhrer L, Betuel H, Seignalet J, Freidel AC, Confavremt C, Billiard M, Daosset J, Cohen D: DNA polymorphism related to HLA-DR2 Dw2 in padents with narcolepsy, lmmunogenetics 22:679, 1985. 16. lnoko H, Ando A, Tsuji K, Matsuki K, Juji T, Honda Y: HLA=D, chain DNA restriction fragments can differentiate between healthy and narcoleptic individuals with HLA-DR2. Immunogenetics 23:126, 1986. 17. Andreas-Zietz A, Keller E, Scholz S, Albert ED, Roth B, Nevsimalova S, Sonka K, Docekal P, lvaskova E, Sdmiz H, Geisler P: DR2-negarive narcolepsy. Lancet i: 684, 1986. 18. Matsuki K. Maeda l-t, Juji T, Inoko H, Ando A, Tsuji K, Honda Y: Taq 1-generated HLA-DQ polymorphism in Japanese padents with narcolepsy, ln~raunogenedcs 27:87, 1988. 19. Marcusson JA, MOiler E: Further studies on psoriasis-associated HLA-Dw/DR.7 using PIT cells primed against a familial psoriasis-linked HLA-haplotype. Tissue Antigens 27:1, 1986.