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Hele tekst

(1)Mesangial cells fail to express CD89. +XPDQ PHVDQJLDO FHOOV LQ FXOWXUH DQG LQ NLGQH\ VHFWLRQV IDLO WR H[SUHVV )F DOSKD UHFHSWRU &'

(2) # 4CNH 9GUVGTJWKU )GT XCP <CPFDGTIGP 0KEQNG #/ 8GTJCIGP 0ICKUCJ -NCT /QJCOCF/QJCOGF4&CJCCPF%GGUXCP-QQVGP &GRCTVOGPVQH0GRJTQNQI[.GKFGP7PKXGTUKV[/GFKECN%GPVGT.GKFGP6JG0GVJGTNCPFU. 6800$5< 6JGOGEJCPKUOQHFGRQUKVKQPQH+I#KP VJG TGPCN OGUCPIKWO KP RTKOCT[ +I# PGRJTQRCVJ[ KU RQQTN[ WPFGTUVQQF +V JCU DGGP UWIIGUVGF VJCV OGODTCPG TG EGRVQTUHQT+I#QPOGUCPIKCNEGNNU /% QH VJG MKFPG[ OC[ DG KPXQNXGF 6Q QD VCKP OQTG KPUKIJV KP VJG QEEWTTGPEG QH VJG O[GNQKF TGEGRVQT HQT +I# %& QP/%DQVJKPUKVWCPFKPEWNVWTGTCD DKV CPF IQCV RQN[ENQPCN CPVKDQFKGU CPF OQWUG OQPQENQPCN CPVKDQF[ CICKPUV TG EQODKPCPV %& YGTG TCKUGF -KFPG[ UGEVKQPU HTQO HKXG EQPVTQN UWDLGEVU CPF HKXG RCVKGPVU YKVJ RTKOCT[ +I#PG RJTQRCVJ[ HCKNGF VQ DG RQUKVKXG HQT %& KP VJG OGUCPIKWO WUKPI QWT RQN[ENQPCN CPF OQPQENQPCN CPVKDQFKGU #NUQ HKXG RTKOCT[ JWOCP /% EWNVWTGU CUUGUUGF HQT %& GZRTGUUKQP UJQYGF PQ RTQVGKP GZRTGUUKQP QH %& (WT. VJGTOQTG TGXGTUG VTCPUETKRVKQP2%4 HCKNGF VQ FGVGEV O40# GZRTGUUKQP QH %&KPVJGEWNVWTGF/%+VYCUFGO QPUVTCVGF VJCV CNN HKXG JWOCP RTKOCT[ /% DQWPF JWOCP +I# KP C FQUGFG RGPFGPV OCPPGT YJKEJ YCU PQV KPJKDK VCDNG D[ DNQEMKPI OQPQENQPCN CPVK %& CPVKDQF[ /[ +P EQPVTCUV DKPFKPI QH +I# VQ 7 EGNNU YCU DNQEMGF GHHKEKGPVN[ D[ /[ (KPCNN[ KPEWDCVKQP QH JWOCP /% YKVJ GKVJGT JWOCP QT TCV +I# NGF VQ KPETGCUGF KPVGTNGWMKP RTQFWEVKQP YJGTGCU QPN[ JWOCP +I# DWV PQV TCV +I# YCU CDNG VQ DKPF VQ JWOCP %& 6JGTGHQTG KV KU EQPENWFGF VJCV JWOCP /% FQ PQV GZRTGUU %& VQ C UKIPKHKECPV GZVGPV 6JGUG TGUWNVU UVTQPIN[ UWIIGUV VJCV DKPFKPIQH+I#VQJWOCP/%QEEWTUXKC CP +I# TGEGRVQT FKUVKPEV HTQO %&. #. Drs Westerhuis and van Zandbergen contributed equally to this study. J Am Soc Nephrol 10:770, 1999. 95.

(3) Chapter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α 60(α 2TGXKQWU TGUWNVU JCXG UJQYP VJCV +I# DKPFU VQ JWOCP/% JQYGXGTPQURGEKHKETGEGRVQTEQWNFDGKFGPVKHKGF 6JG&0#UGSWGPEGQHCTGEGRVQTHQTVJG(EVCKNQH+I#GZRTGUUGFQPO[GNQKFEGNNU YCU RWDNKUJGF GCTNKGT (WTVJGT KPXGUVKICVKQPU HQWPF GZRTGUUKQP QH VJKU +I# TGEGRVQTFGUKIPCVGF(EαTGEGRVQTQT%&QPVJGUWTHCEGQHPGWVTQRJKNU GQUKPQRJKNU CPFEGNNUQHVJGOQPQE[VGOCETQRJCIGNKPGCIG 8GT[TGEGPVN[O40#GZRTGUUKQPQHVJG(ETGEGRVQT%&JCUDGGPUWIIGUVGF QPJWOCP/% #EVKXCVKQPQHJWOCP/%YKVJ+.QT60(αNGFVQKPETGCUGF DKPFKPI QH +I# CEEQORCPKGF D[ CP KPETGCUG QH O40# GZRTGUUKQP QH %& *QYGXGT PQ GXKFGPEG YCU RTQXKFGF HQT VJG FKTGEV KPXQNXGOGPV KP VJG DKPFKPI QH +I# VQ VJGUG EGNNU XKC %& 6YQ CFFKVKQPCN UVWFKGU UJQYGF CP KPETGCUG QH O40# GZRTGUUKQP QH OQPQE[VG EJGOQCVVTCEVCPV RTQVGKP +. PWENGCT HCEVQT κ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µIONUVTGRVQO[EKP CNNHTQO.KHG6GEJPQNQIKGU$TGFC6JG0GVJGTNCPFU . 96.

(4) Mesangial cells fail to express CD89. 6JGOWTKPG$EGNNNKPG++# YCUEWNVWTGFKP42/+UWRRNGOGPVGF YKVJ(%5++#YCUVTCPUHGEVGFYKVJ%&E&0#CPFUVCDNGEGNNUWTHCEG GZRTGUUKQPYCUOCKPVCKPGFD[EQVTCPUHGEVKQPQHJWOCPγ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µ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µOQN COKPQRVGTKP µ/ VJ[OKFKPG µ/ 7.RGPKEKNNKPCPF µ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

(5) Chapter 6. 3RO\FORQDODQWLERGLHV #TCDDKVCPFCIQCVYGTGKOOWPK\GFYKVJµ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µION HQT J YGTG WUGF 6QVCN EGNNWNCT 40# YCU KUQNCVGF HTQO Z EGNNU WUKPI 40#\QN $ %KPPC$KQVGEZ *QWUVQP 6: CEEQTFKPIVQOCPWHCEVWTGTNUKPUVTWEVKQPU (KZGFCOQWPVUQHVQVCNEGNNWNCT40#. µI YGTGTGXGTUGVTCPUETKDGFKPVQE&0#D[QNKIQ F6 RTKOKPIWUKPI/QNQPG[ OWTKPGNGWMGOKCXKTWUTGXGTUGVTCPUETKRVCUG .KHG6GEJPQNQIKGU 6JGCORNKHKECVKQPQHE&0#D[2%4YCURGTHQTOGFWUKPIVJGRTKOGTUCUUJQYP KP (KIWTG 9G VGUVGF UGXGP FKHHGTGPV EQODKPCVKQPU QH VJG CDQXGFGUETKDGF HQTYCTFCPFTGXGTUGRTKOGTUCUUJQYP(WTVJGTOQTGYGRGTHQTOGFCPGUVGF2%4KP 98.

(6) Mesangial cells fail to express CD89. YJKEJYGHKTUVWUGFVJGHQTYCTFRTKOGT DRVQ EQODKPGFYKVJVJGTGXGTUG RTKOGT DRVQ 6JGRTQFWEVQHVJKU2%4YCUEJGEMGFQPCICTQUGIGNCPF VJGPCPFµIQHVJG2%4RTQFWEVYGTGCICKPCORNKHKGFD[2%4WUKPI VJG HQTYCTF RTKOGT DR VQ CPF VJG TGXGTUG RTKOGT DR VQ 2%4CORNKHKECVKQPYCURGTHQTOGFWPFGTUVCPFCTFEQPFKVKQPU O/-%NO/ 6TKU*%N R* OOQN. /I%N OION DQXKPG UGTWO CNDWOKP O/ F062 ROQN QH GCEJ RTKOGT CPF 7 QH 6CS RQN[OGTCUG 2GTMKP 'NOGT 0QTYCNM%6 D[E[ENGUQHVJGHQNNQYKPIUEJGOGOKPCV°%OKPCV °%OKP°%HQNNQYGFD[OKPQHRTKOGTGZVGPUKQPCV°%/I%N EQPEGPVTCVKQPUQHOOQN.YCUHQWPFVQDGUWRGTKQTEQORCTGFYKVJQT OOQN. HQT VJG CORNKHKECVKQP QH %& 2%4 RTQFWEVU YGTG CPCN[\GF QP C CICTQUG IGN EQPVCKPKPI GVJKFKWO DTQOKFG 4GUWNVU YGTG TGIKUVGTGF WUKPI C FKIKVCN ECOGTC 'CING G[G 5VTCVCIGPG 5CP &KGIQ %# CPF HQT TGCUQP QH ENCTKV[ VJG KOCIGUYGTGDNCEMYJKVGKPXGTVGF Figure 1. Gene of CD89 and the used primers. At the top, the gene of CD89 is shown containing five exons: S1, S2, extracellular-1 (EC1), EC2, and TM/C (transmembrane/ cytoplasmic). The six different primers shown at the bottom are indicated by an arrow below the CD89 gene to illustrate their location. Three forward and three reverse primers were used in different combinations. Primers for GAPDH were used as positive control.. 5’. 34 36. 291. 288. 215. bp. S1 S2. EC1. EC2. TM/C. 3’. 1. Primers No.. 2. 3.. 4.. Primer sequence 5’- 3’. GAPDH ccAcccATggcAAATTccATg cTAgAcggcAggTcAggTcc GAPDH for CD89: ccTccTgTgTcTTgTgcTcT 1. gAgGATTcAggcAcAggAAg 2. ccATgccTTTcATATcTgcc 3. TcTcTccTggcATcAAcAcc 4. gTTgTAccAAccgTAgcAcc 5. cTTgcAgAcAcTTggTgTTc 6.. 5.. 6.. Primer position forward/ reverse 185 - 205 762 - 781. forward reverse. 60 - 79 90 - 109 118 - 138 460 - 441 633 - 614 900 - 881. forward forward forward reverse reverse reverse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

(7) Chapter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µION #UCRQUKVKXGEQPVTQN9YCUWUGF. EWNVWTG UWRGTPCVCPV +I)C OQWUG O#D #HVGT KPEWDCVKQP EGNNU YGTG YCUJGF VYKEG YKVJ (#%5 DWHHGT CPF KPEWDCVGF HQT JQWT YKVJ IQCV CPVKOQWUG +I)RJ[EQGT[VJTKP 2' RQN[ENQPCN CPVKUGTWO IQCV CPVKTCDDKV +I)2' RQN[ENQPCN CPVKUGTWOTCDDKVCPVKIQCVK(+6%CPFIQCVCPVKOQWUG+I)2'RQN[ENQPCNCPVKUGTWO TGURGEVKXGN[ CNNHTQO&CMQ #NNUVCKPKPIRTQEGFWTGUYGTGRGTHQTOGFCV°% 6JGDKPFKPIQHJWOCP+I#VQ7CPF /% YCU CPCN[\GF CU HQNNQYU EGNNU YGTG YCUJGF VYKEG YKVJ (#%5 DWHHGT CPF KPEWDCVGF HQT J YKVJ CPF µIONRWTKHKGF+I##HVGTKPEWDCVKQPVJGEGNNUYGTGYCUJGFCPFDQWPF+I# YCU FGVGEVGF D[ KPEWDCVKQP YKVJ OQWUG OQPQENQPCN CPVKJWOCP +I# CPVKDQF[. ' CPF UWDUGSWGPVN[ YKVJ 2'NCDGNGF IQCV CPVKOQWUG +I) RQN[ENQPCN CPVKUGTWO 6Q FGOQPUVTCVG VJG URGEKHKEKV[ QH DKPFKPI QH +I# VQ EGNNWNCT %& UVWFKGU YGTGRGTHQTOGFKPYJKEJ7CPF/%YGTGKPEWDCVGFYKVJ+I#KPVJGRTGUGPEG CPFCDUGPEGQHVJG%&DNQEMKPIOQWUGO#D/[ #PKUQV[RGOCVEJGF KTTGNGXCPVOQWUGO#DUGTXGFCUCEQPVTQNHQT/[ 6QFGVGTOKPGVJGURGEKHKEKV[QHTCVCPFJWOCP+I#HQTJWOCP%&++# %&VTCPUHGEVGFEGNNUYGTGVGUVGFHQTDKPFKPIQHJWOCPCPFTCV+I##VQVCNQH. 100.

(8) Mesangial cells fail to express CD89. µIONJWOCP+I#QTTCVFKOGTKE+I#YCUCFFGFVQVJGEGNNUHQTJ6JGP EGNNU YGTG YCUJGF CPF DQWPF +I# YCU FGVGEVGF D[ KPEWDCVKQP YKVJ OQWUG OQPQENQPCNCPVKJWOCP+I#CPVKDQF[ ' DKQVKPQTTCDDKVRQN[ENQPCNCPVKTCV+I# DKQVKP TGURGEVKXGN[ #HVGT YCUJKPI EGNNU YGTG KPEWDCVGF HQT J YKVJ UVTGRVCXKFKP 2' &CMQ 6JG RGTEGPVCIG RQUKVKXG EGNNU YGTG WUGF KP CNN (#%5GZRGTKOGPVU CU C OGCUWTGHQT%&GZRTGUUKQPCPF+I#DKPFKPI. A77. relative cell number. Figure 2. Fluorescence-activated cell sorter (FACS) analyses of CD89 expression on U937 cells by six mouse monoclonal antibodies (mAb). A77, a defined Ms mAb anti-CD89, and five new mouse mAb anti-CD89 (2E6, 2D11, 7G4, 2H8, and 7D7) were tested on FACS using CD89-expressing U937 cells. Broken lines show the staining of the cells by an isotype-matched control antibody: the solid lines show the staining with the anti-CD89 mAb. The dotted lines in all panels indicate the mean staining obtained with A77.. 100 101 102 103 104. 7G4. 100 101 102 103 104. 2E6. 2D11. 100 101 102 103 104 100 101. 2H8. 102 103 104. 7D7. 100 101 102 103 104. 100 101. 102 103 104. log. mean fluorescence intensity (MFI). ,/SURGXFWLRQE\KXPDQ0&LQYLWUR. Figure 3. FACS analysis of CD89 expression on U937 cells. Two polyclonal anti-CD89 antibodies (rabbit and goat) were tested on U937 cells. The broken lines show the staining of the cells using a normal control antibody, the solid lines show the staining with the polyclonal antibodies.. relative cell number. 5WDEQPHNWGPV YGNN RNCVGU YKVJ JWOCP /% YGTG YCUJGF VKOGU YKVJ 2$5 CPF EWNVWTGF HQT CPQVJGT J KP &WNDGEEQNU OQFKHKGF 'CINGNU OGFKWO (%5 VQ DTKPI VJG EGNNU VQ C SWKGUEGPV UVCVG #HVGT YCUJKPI VJG EGNNU VJTGG VKOGU YKVJ OGFKWO µI QH FKOGTKE JWOCP CPF TCV +I# YCU CFFGF VQ VTKRNKECVG YGNNU #HVGT J QH KPEWDCVKQP UWRGTPCVCPVU YGTG JCTXGUVGF CPF CUUGUUGF HQT +. RTQFWEVKQP WUKPI VJG +. FGRGPFGPV OWTKPG J[DTKFQOC EGNN NKPG $ 5GTKCN FKNWVKQPU QH JWOCP TGEQODKPCPV +. YGTG WUGF CU Gt-α-CD89 Rb-α-CD89 CUVCPFCTF on U937-cells on U937-cells. 100 101. 102 103 104. 100 101. 102 103 104. MFI. 101.

(9) Chapter 6. 6WDWLVWLFDODQDO\VHV #NNXCNWGUCTGGZRTGUUGFCUOGCPv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igure 4. Western blot analysis for the characterization of the monoclonal and polyclonal anti-CD89 antibodies. Soluble recombinant CD89 was blotted and stained using the five new mouse mAb anti-CD89, A77, and two polyclonal anti-CD89 antibodies. As an example, results using A77, 7D7 and 7G4 are shown. As the negative control, we used an isotype-matched irrelevant mouse mAb. The other monoclonal and polyclonal antibodies were also positive using this method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

(10) Mesangial cells fail to express CD89. ,PPXQRIOXRUHVFHQFHRIFHOOVJURZQRQJODVVFRYHUVOLSV /% HTQO HKXG QH HKXG RTKOCT[ /% EWNVWTGU ITQYP HQT J QP INCUU EQXGTUNKRU TGXGCNGF PQ RQUKVKXG UVCKPKPI HQT %& WUKPI CNN QWT CPVKDQFKGU CPF # (KIWTG 1P VJG QVJGT JCPF %& GZRTGUUKQP QP VJG OQPQE[VKE EGNN NKPG 7 YCU ENGCTN[ RTGUGPV #U GZRGEVGF GZRTGUUKQP QH /*% ENCUU + YCU RQUKVKXG HQT DQVJ 7EGNNUCPF/% (KIWTG . A. D. A. D. B. E. B. E. C. F. C. F. Figure 5. Tissue staining of human kidney and spleen cryostat sections. Cryostat sections of human kidney from patients with IgAN (D through F) and normal spleen (A through C) were stained for MHC-class I (B and E), CD89 (C and F), and control mAb (A and D). For MHC class I staining, W6-32 was used and for CD89 the whole set of our monoclonal and polyclonal antibodies was used. As an example, stainings with one mouse mAb anti-CD89 (7G4) is shown (C and F).. Figure 6. CD89 stainings for mesangial cells (MC) and U937 cells grown on glass coverslips. Human MC (D through F) derived from five different individuals and U937 cells (A through C) were stained for MHC class I (B and E) and CD89 (C and F). For MHC class I staining, W6-32 was used and for CD89 the whole set of our new monoclonal and polyclonal antibodies was used. As an example, a staining with mouse mAb 7G4 is shown (C and F). A and D show the negative control stained with an isotype-matched antibody.. 103.

(11) Chapter 6. )$&6DQDO\VLVIRU&'H[SUHVVLRQ 7CPF/%YGTGCNUQCPCN[\GFHQT%&RTQVGKPGZRTGUUKQPD[(#%5CPCN[UKU #NVJQWIJ7EGNNUYGTGUVTQPIN[RQUKVKXGHQT%&CNNQHVJGHKXGRTKOCT[/% YGTGEQORNGVGN[PGICVKXGYKVJCNNOQPQENQPCNCPFRQN[ENQPCNCPVKDQFKGU (KIWTG +PEQPVTCUV/*%ENCUU+GZRTGUUKQPYCUENGCTN[RQUKVKXGHQTDQVJ7CPF/% Rb-poAbα-CD89. Ms-mAb-αCD89. U937. mAb-α-MHC class I. 100 101 102 103 104 100 101. 102 103 104 100 101. MC 1. 102 103 104 100 101 102 103 104. MC2. 102 103 104. relative cell number. 100 101 102 103 104 100 101. Figure 7. FACS analysis of CD89 expression on U937 and MC. CD89 expression was analyzed with five mouse mAb anti-CD89 and two polyclonal antibodies directed against CD89. Histograms of one mono-clonal and one polyclonal antibody are shown as representative examples for two of the five examined primary MC cultures. As a positive control MHC class 1 detection by W6-32 is shown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µION PQ O40# HTQO %& YCU HQWPF (KPCNN[YGRGTHQTOGFC PGUVGF 2%4 WUKPI VJTGG FKHHGTGPV EQPEGPVTCVKQPU CPF µN QH VJG RTQFWEV QH C HKTUV 462%4 KP C UGEQPF 2%4 YKVJ KPVGTPCN RTKOGTU 6JG %&GZRTGUUKPI EQPVTQN EGNNU YGTG RQUKVKXG KP CNN ECUGU YJGTGCU O40# KUQNCVGF HTQO JWOCP /% HCKNGF VQ DG RQUKVKXG HQT %& GXGP CHVGT VJKU UGEQPFTQWPFQHCORNKHKECVKQP (KIWTG MFI. 104.

(12) Mesangial cells fail to express CD89. %LQGLQJRIKXPDQ,J$WR0& +PEWDCVKQPQH/%YKVJ+I#CPFUWDUGSWGPVCPCN[UKUD[(#%5TGXGCNGFVJCVCNNHKXG /%DQWPFJWOCP+I#YKVJCOGCPQH±RQUKVKXGEGNNU TCPIKPIHTQO VQ 6Q FGVGTOKPG YJGVJGT VJG DKPFKPI QH +I# VQ VJG /% YCU OGFKCVGFD[%&/%YGTGGZRQUGFVQ+I#KPVJGRTGUGPEGCPFCDUGPEGQHVJG O#D CPVK%& /[ CPF UWDUGSWGPVN[ CUUGUUGF HQT DKPFKPI QH +I# /[ FKF PQVTGFWEGVJGDKPFKPIQH+I#VQJWOCP/% ± DWVKVKPJKDKVGFVJG DKPFKPI QH +I# VQ 7 EGNNU D[ (KIWTG 6JG 7 EGNNU YKVJQWV RTGKPEWDCVKQP YKVJ +I# UJQYGF C DCUCN /(+ QH %GNNU KPEWDCVGF YKVJ +I# KPETGCUGF VJGKT OGCP HNWQTGUEGPEG KPVGPUKV[ /(+ VQ CPF VJQUG EGNNU RTGKPEWDCVGF YKVJ VJG DNQEMKPI CPVK%& CPVKDQF[ /[ UJQYGF OKPKOCN DKPFKPIQH+I#KPFKECVGFD[CP/(+QH Figure 8. Reverse Transcription (RT)-PCR analysis to detect CD89 mRNA expression. (A) Results of RT-PCR obtained with eight different primer sets. None of the primer combinations for CD89 was able to detect mRNA of this receptor in five of five primary MC cultures, whereas only primer combination 3+6 was negative for U937- and IIA1.6-CD89-transfected cells. All other combinations showed positive bands. Control primers detec-ting mRNA expression of GAPDH was positive for all the examined cell lines. As a negative control, water was used. (B) Three examples of a primer combination as well as the result of the nested RT-PCR for CD89. GAPDH control is shown at the top.. A Primer set. 2+5/ 1+4 1+5 2+4 2+5 3+4 3+5 3+6 3+4 +. +. +. +. +. +. -. +. IIA1.6 CD89 +. +. +. +. +. +. -. +. -. -. -. -. -. -. -. -. U937. 5hu MC. B. H2O U937 IIA1.6 MC1. MC2. MC3. MC4. MC5. GAPDH CD89 primer 3 and 5 CD89 primer 2 and 4 CD89 primer 2 and 5 Nested RT-PCR 2+5/ 3+4. 3URGXFWLRQRI,/DIWHULQFXEDWLRQRI0&UDWDQGKXPDQ,J$ 6QFGVGTOKPGVJGGHHGEVQHFKOGTKETCVCPFJWOCP+I#/% YGTG KPEWDCVGF YKVJ µIQHTCVQTJWOCP+I#CPFCUUGUUGFHQT+.RTQFWEVKQP6JGDCUCNRTQFWE VKQP YCU ± 7 +. RGT EGNNU +PEWDCVKQP QH /% YKVJ µI QH 105.

(13) Chapter 6. JWOCP+I#TGUWNVGFKPGPJCPEGOGPV+.RTQFWEVKQPVQ±7+.RGT EGNNU 2 +PVGTGUVKPIN[ TCV +I# CNUQ KPFWEGF CP GPJCPEGOGPV QH +. CPF TGUWNVGF KP RTQFWEVKQPQH control IgA IgA + My43 ± 7 +. RGT EGNNU 2 SS $QVJ TGUWNVU CTG SSC C 1% 17,7% 17,5 % UKIPKHKECPVN[ JKIJGT EQORCTGF YKVJ /% EWNVWTGF KP OGFKWO CNQPG (KIWTG Figure 9. Effect of My43 on the binding of IgA1 to U937, and MC. (A) Contour pro1.6% 3.8 % 43.8% files of MC incubated without (left panel) and with (middle panel) MFI human IgA. In this case, 17.7% positive cells were observed. The panel on the right shows the binding of IgA to MC in the presence of My43. Similar results were found using four other primary MC. (B) Contour profiles of U937 cells showing 43.8% positive cells with a mean fluorescence intensity (MFI) of 28.2 after incubation of U937 cells with IgA (middle panel). In the presence of My43, a reduction of IgA-binding to 3.8% respective a mean fluorescence intensity (MFI) of 5.81 was observed. U937 incubated without IgA are shown in the left panel (MFI) 3.05). Comparable results were obtained in three different experiments.. SS SSC C. )$&6 DQDO\VLV RI ELQGLQJ RI KXPDQ ,J$ DQG UDW GLPHULF ,J$ WR &'WUDQVIHFWHGFHOOV. IL-6 (units/105 cells). 6QVGUVVJGURGEKHKEKV[QHJWOCP+I#HQT%&(#%5CPCN[UKUWUKPI++#%& VTCPUHGEVGF EGNNU GZRTGUUKPI TGEQODKPCPV JWOCP %& QP VJGKT UWTHCEG YCU RGTHQTOGF #HVGT KPEWDCVKQP ++# human rat %&VTCPUHGEVGF EGNNU YKVJ JWOCP CPF 5000 TCV +I# C FKHHGTGPEG KP DKPFKPI YCU 4000 HQWPF 3000 2000 1000 0 medium. 100. 100. concentration mg/ml (IgA). 106. Figure 10 IgA-induced enhancement of interleukin-6 (IL-6) production by human MC. The effect of human (100 µg/ml) and rat IgA (100 µg/ml) on production of IL-6 by human MC compared to MC incubated in medium alone is shown. The results are means ± SD of three experiments..

(14) Mesangial cells fail to express CD89. relative cell number. human IgA. 100 101. rat IgA. 102 103 104. 100 101. MFI. 102 103 104. Figure 11. FACS analysis of the binding of human and rat IgA to IIA1.6-CD89-transfected cells. IIA1.6 CD89-transfected cells were incubated with 100 µg of human or rat IgA, respectively. Almost 100 % of the CD89-transfected cells bound human IgA (left histogram) in contrast to the cells incubated with rat IgA (right histogram). Control stainings without IgA are depicted as dotted lines. On the other hand, rat IgA was able to bind to human MC (data not shown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

(15) Chapter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α CPF KPVGTHGTQPγ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

(16) Mesangial cells fail to express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

(17) Chapter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

(18) Mesangial cells fail to express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

(19) Chapter 6 RWTKHKGFTCVJGRCVKECUKCNQIN[EQRTQVGKPTGEGRVQTU CPF FKUVKPIWKUJGU VYQ TGEGRVQT RQRWNCVKQPU , $KQN%JGO. 112. 5VQEMGTV 4 , 6JG CUKCNQIN[EQRTQ VGKP TGEGRVQT TGNCVKQPUJKRU DGVYGGP UVTWEVWTG HWPEVKQPCPFGZRTGUUKQP2J[UKQN4GX.

(20)

QH VJG MKFPG[ OC[ DG KPXQNXGF 6Q QD VCKP OQTG KPUKIJV KP VJG QEEWTTGPEG QH VJG O[GNQKF TGEGRVQT HQT +I# %&amp

QH VJG MKFPG[ OC[ DG KPXQNXGF 6Q QD VCKP OQTG KPUKIJV KP VJG QEEWTTGPEG QH VJG O[GNQKF TGEGRVQT HQT +I# %&amp