(1)Reduced binding of mIgA from IgAN patients to CD89

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(1)Reduced binding of mIgA from IgAN patients to CD89. 5HGXFHGELQGLQJRILPPXQRJOREXOLQ$ ,J$

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(10) Chapter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α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

(11) Reduced binding of mIgA from IgAN patients to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γEJCKP E&0# CU FGUETKDGF RTGXKQWUN[ 6JG %&VTCPUHGEVCPVU YGTG ITQYP KP VJG UCOG OGFKWOUWRRNGOGPVGFYKVJIGPGVKEKP )OION.KHG6GEJPQNQIKGU CPF OGVJQVTGZCVG /6:OOQNN2JCTOCEJGOKG*CCTNGO6JG0GVJGTNCPFU 6JG JWOCPOQPQE[VKENKMGEGNNNKPG7YCUEWNVWTGFKP42/+UWRRNGOGPVGF YKVJJGCVKPCEVKXCVGF(%5 'HWHFWLRQRI&' %&VTCPUHGEVGF CPF EQPVTQN EGNNU YGTG VGUVGF HQT %& GZRTGUUKQP D[ (#%5 CPCN[UKU WUKPI %& O#D # %GNNU Z YGTG YCUJGF VYKEG YKVJ (#%5 DWHHGT 2$5$5# CPF KPEWDCVGF HQT J CV u% YKVJ # EWNVWTG UWRGTPCVCPVFKNWVGFKP(#%5DWHHGT(QNNQYKPIKPEWDCVKQPEGNNUYGTGYCUJGFVYKEG. (#%5 DWHHGT CPF KPEWDCVGF HQT J YKVJ C IQCV CPVKOQWUG +I)2' RQN[ENQPCN CPVKUGTWO FKNWVGF KP (#%5 DWHHGT #HVGT YCUJKPI VYKEG YKVJ (#%5 DWHHGT EGNNU YGTG HKZGF KP RCTCHQTOCNFGJ[FG KP 2$5 CPF CPCN[\GF QP C (#%5ECP $GEVQP &KEMKPUQP5CP,QUG%# &CVCCESWKUKVKQPCPFCPCN[UKUYGTGEQPFWEVGFYKVJ.[UKU ++ $GEVQP&KEMKPUQP . 83.

(12) Chapter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u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

(13) Reduced binding of mIgA from IgAN patients to CD89. VQVCN+I#D['.+5#CPFVJGDKPFKPIQH+I#VQ%&VTCPUHGEVGF++#EGNNUYCU VGUVGFCUFGUETKDGFCDQXG %LQGLQJ RI ,J$ ,J$ DQG ,J$

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(15) Chapter 5. 7UKPIVJKUU[UVGOYGEQWNFENGCTN[FGVGEVDKPFKPIVQ%&VTCPUHGEVGFEGNNUQHRW TKHKGFR+I#CPFF+I#DWVCNUQDKPFKPIQHO+I#YCUENGCTN[XKUKDNG (KIWTG A. Relative Cell Number. IIA1.6 Control cells. IIA1.6 CD89-transfected cells. A77 100 101 102 103 104. 100 101 102 103 104. pIgA 100 101 102 103 104. 100 101 102 103 104. Log Fluorescence Intensity. Figure 1. FACS analysis of IgA binding to CD89-transfected IIA1.6 cells A. FACS histograms of IIA1.6 control cells (left) and CD89-transfected IIA1.6 cells (right) are shown. Surface expression of CD89 was detected with mAb A77(anti-CD89) (top) and binding of polymeric IgA (50µg/ml) to CD89 was detected with mAb 4E8 (antiIgA) followed by Gt-anti-Ms IgG1-PE (bottom). Filled histograms represents the conjugate controls, open histograms represent either CD89 expression (top) or pIgA binding (bottom). B. FACS histograms of CD89-transfected IIA1.6 cells are shown. IgA binding was detected as described above, using aggregated IgA (50µg/ml) in the presence of an irrelevant IgM mAb (left) or in the presence of IgM mAb MY43 (anti-CD89) (right).. 0GZV YG CUUGUUGF VJG DKPFKPI VQ %&VTCPUHGEVGF++#EGNNUQH+I# Control mAb My43 FKTGEVN[ HTQO YJQNG UGTWO +PEWDC VKQPQH%&VTCPUHGEVGF++#EGNNU aIgA YKVJ XCT[KPI FKNWVKQPU QH UGTWO TGUWNVGF KP C FQUGFGRGPFGPV DKPFKPI 100 101 102 103 104 QH +I# VQ VJG EGNNU 0Q FGVGEVCDNG 100 101 102 103 104 Log Fluorescence Intensity DKPFKPI VQ PQPVTCPUHGEVGF ++# EGNNUYCUQDUGTXGF9JGPYG UVWFKGF QVJGT KUQV[RGU YG EQWNF PQV FGOQPUVTCVG CP[ DKPFKPI QH +I/ QT +I) VQ %& VTCPUHGEVGFEGNNU FCVCPQVUJQYP 6JGDKPFKPIQH+I#YCUDGNKPGCTKPCFKNWVKQP TCPIG QH UGTWO HTQO VQ . FCVCPQVUJQYP 200 IIA1.6 CD89-transfected cells. Relative Cell Number. B. Gt-a-Hu-IgA Ms-a-Hu-IgA(4E8). MFI. 150 Figure 2. Detection of IgA binding to CD89transfected cells with IgA specific antibodies. PIgA, dIgA and mIgA were purified from 100 sera of a myeloma patient and tested for binding to CD89 transfected cells. Binding of 25 50 µg/ml of pIgA, dIgA and mIgA was analyzed with either Gt-α-Hu-IgA polyclonal detected 0 with PE conjugated Rb-α-Gt polyclonal (open pIgA dIgA mIgA bars), or Ms-α-Hu-IgA mAb (4E8) detected with PE conjugated Gt-α-Ms IgG1 polyclonal (filled bars). Indicated is the MFI, as a measure for binding, which is calculated as described in patients and methods.. 86.

(16) Reduced binding of mIgA from IgAN patients to CD89. %LQGLQJRI,J$IURPVHUDWR&'WUDQVIHFWHG,,$FHOOV. MFI. 6QCUUGUUVJGDKPFKPIVQ%&VTCPUHGEVGF++#EGNNUQH+I#FKTGEVN[HTQOYJQNG UGTWO UGTC HTQO RCVKGPVU YKVJ +I#0 CPF EQPVTQNU YGTG VGUVGF HQT DKPFKPI VQ %&VTCPUHGEVGF++#EGNNUCVCHKZGFFKNWVKQPQH 6CDNG #UUJQYPHQT RWTKHKGF+I#RTGRCTCVKQPUCNUQUGTWO+I#FKFPQVUJQYDKPFKPIVQPQPVTCPUHGEVGF EGNNU FCVC PQV UJQYP *QYGXGT KP CNN ECUGU Table 1. IgA binding to CD89-transfected cells DKPFKPI VQ %&VTCPU HGEVGF ++# EGNNU QH IgA1 IgA2 +I# FKTGEVN[ HTQO YJQNG UGTWO YCU GXKFGPV 2C Controls 69.59 ± 2.7 25.65 ± 2.3 VKGPVU YKVJ +I#0 JCF UKIPKHKECPVN[ JKIJGT EQP Patients 70.70 ± 3.4 31.30 ± 2.3 EGPVTCVKQPU QH +I#. v OION Binding of serum IgA in MFI ± SEM. of patients with IgAN (n=23) and controls (n=17) to CD89-transfected cells. Binding of IgA1 or IgA2 was determined as XU v described in the patients and methods section. OION2 CPF +I# v OION XU v OION 2 KP VJGKT UGTC YJKNGPQUKIPKHKECPVFKHHGTGPEGYCUHQWPFHQT+I# vOIONXU vOION2 +PVGTGUVKPIN[VJGJKIJGTUGTWO+I#EQPEGPVTCVKQPUKP RCVKGPVUYKVJ+I#0FKFPQVTGUWNVKPGPJCPEGFDKPFKPIVQ%&VTCPUHGEVGF++# EGNNU CU EQORCTGF VQ EQPVTQNU. (KIWTG 6JGDKPFKPIQHVJG+I# 100 UWDENCUUGU+I#CPF+I#UJQYGF PQ FKHHGTGPEG DGVYGGP RCVKGPVU 75 CPFEQPVTQNU 6CDNG 50. 25. 0. Control (n=17) IgAN (n=23). Figure 3. Binding to CD89-transfected IIA1.6 cells of IgA directly from serum of IgAN patients and controls. Sera of patients (closed circles) with IgAN (n=23) and controls (open circles)(n=17) were tested for binding to CD89-transfected IIA1.6 cells. Binding of IgA was detected as described in figure 1. Indicated is the MFI, as a measure for binding, which is calculated as described in patients and methods.. 6L]HIUDFWLRQDWLRQRIVHUXP,J$ 9GFGOQPUVTCVGFRTGXKQWUN[VJCVRWTKHKGFR+I#DKPFUDGVVGTVQ%&VJCPO+I#. 6JGTGHQTGVJGCOQWPVQH+I#DQWPFVQ%&KUPQVQPN[FGVGTOKPGFD[EQP 87.

(17) Chapter 5. EGPVTCVKQPUQH+I#CUFGUETKDGFCDQXGDWVCNUQD[VJGEQPVGPVCPFTCVKQ QH RQN[ OGTKEFKOGTKECPFOQPQOGTKE+I#KPVJGUGTC+PQTFGTVQQDVCKPKPUKIJVKPVJGGH HGEVQHUK\GQPVJGDKPFKPIVQ%&QH+I#HTQORCVKGPVUYKVJ+I#0UGTCQHRC VKGPVU CPF EQPVTQNU YGTG A HKTUV HTCEVKQPCVGF D[ IGN Control HKNVTCVKQP EJTQOCVQITC 500 75 IgA in µg/ml RJ[ (KIWTG 50 MFI. IgA in µg/ml. MFI. 250. 25. 0. 0 pIgA dIgA. mIgA. Fractions. B. Figure 4. Fractionation of IgAN and control serum on S300 Sephacryl. Sera of controls (A) with IgAN and patients (B) were fractionated by size on a S300 Sephacryl column. Fractions were test-ed for IgA concentrations (closed squares) and asses-sed for binding to CD89-transfected IIA1.6 cells (open circles). One representative example from each group, which contains 6 individuals, is shown.. IgAN patient 1500. 150. 6JG RGTEGPVCIGU QH RQN[ OGTKE FKOGTKE CPF OQ MFI PQOGTKE +I# ECNEWNCVGF 1000 100 HTQO VJG +I# EQPEGP VTCVKQP KP VJG UGTC UCORNGU CHVGT HTCEVKQP 500 50 CVKQP YGTG PQV FKHHGTGPV DGVYGGP RCVKGPVU CPF EQPVTQNU 6CDNG 0 0 9JGP VJG DKPFKPI QH VJG pIgA dIgA mIgA +I# EQPVCKPKPI HTCEVKQPU Fractions YCU CUUGUUGF QP %& VTCPUHGEVGF ++# EGNNU R+I# CPF F+I# EQPVCKPKPI HTCEVKQPU QH RCVKGPVU YKVJ +I#0 CPF EQPVTQNU UJQYGF UKIPKHKECPVN[ JKIJGT /(+ DKPFKPI XCNWGU EQORCTGF VQ O+I# EQPVCKPKPI HTCEVKQPU +PVGTGUVKPIN[ O+I# HTQO RCVKGPVU YKVJ +I#0 UJQYGF TGFWEGF /(+ XCNWGU EQORCTGFVQOQPQOGTKE+I#HTQOEQPVTQNUGXGPVJQWIJVJGO+I#EQPEGPVTCVKQPU KPVJGVGUVGFHTCEVKQPUQHVJGRCVKGPVUYKVJ+I#0YCU`HQNFJKIJGTCUEQORCTGF VQVJGEQPVTQNU (KIWTG 88. MFI. IgA in µg/ml. IgA in µg/ml.

(18) Reduced binding of mIgA from IgAN patients to CD89. %LQGLQJRIS,J$G,J$DQGP,J$WR&'WUDQVIHFWHGFHOOV 0GZVYGCUUGUUGFDKPFKPIVQ%&VTCPUHGEVGF++#EGNNUQHRGCMHTCEVKQPUEQP VCKPKPI R+I# F+I# CPF O+I# 6JGUG HTCEVKQPU Table 2. IgA size distribution from patients with IgAN and controls EJQUGP UJQYGF PQ QXGTNCR KP UK\G CU FG pIgA dIgA mIgA VGTOKPGFD[ITCFKGPVIGN CPCN[UKU. FCVC PQV Controls 13.36 ± 1.9 28.92 ± 1.4 57.72 ± 2.0 UJQYP (KTUV VJGUG HTCE VKQPU YGTG VGUVGF CV C Patients 9.70 ± 1.0 27.92 ± 2.1 62.38 ± 2.9 EQPEGPVTCVKQP QH zION #V VJGUG HKZGF Distribution of polymeric, dimeric and monomeric IgA from fractionated sera of EQPEGPVTCVKQPUO+I#QH patients with IgAN (n=6) and controls (n=6) given as percentages ± SEM. RCVKGPVU YKVJ +I#0 UJQ YGFCUVTQPIN[FGETGCUGFDKPFKPIVQ%&CUEQORCTGFVQEQPVTQNU (KIWTG# A. B IgAN. 30 Control IgAN. *. 20 MFI. Relative Cell Number. Monomeric IgA. Control. 10 100 101 102 103 104. *. 100 101 102 103 104. Log Fluorescence Intensity. 0 0. 0.25. 2.5. 25. 250. MFI. Figure 5. Binding of monomeric and polyIgA in µg/ml C meric IgA from controls and IgAN patients Polymeric IgA to CD89-transfected IIA1.6 cells A. The 50 peak fractions of the gelfiltration columns Control representing monomeric IgA were tested for IgAN 40 binding to CD89-transfected cells at a 30 concentration of 25 µg/ml. From each group (n=6) one representative flow cytometry 20 histogram is shown: controls (left), patients 10 with IgAN (right). Filled histograms represent the conjugate controls, open histograms 0 represent the binding of mIgA to CD890 0.25 2.5 25 250 transfected cells. B,C. The peak fractions of IgA in µg/ml the gelfiltration columns representing monomeric (B) and polymeric (C) IgA from sera of patients (closed circles) with IgAN (n=6) and controls (open circles) (n=6) were tested for binding to CD89-transfected IIA1.6 cells. IgA was tested in a dose response with the fixed concentration of resp. 0.25, 2.5, 25 µg/ml. Mean ± SEM are depicted, (*:P<0.05). 89.

(19) Chapter 5. 9JGPVGUVGFKPCFQUGTGURQPUGVJGTGYCUCUKIPKHKECPVN[ 2 NQYGTDKPFKPI. /(+ QHO+I#VQ%&HTQORCVKGPVUYKVJ+I#0CUEQORCTGFVQEQPVTQNU (KIWTG $ $KPFKPIVQ %&VTCPUHGEVGF ++# EGNNU QH F+I# CPF R+I# QH RCVKGPVU YCU PQVUKIPKHKECPVN[TGFWEGFCUEQORCTGFVQF+I#CPFR+I#QHEQPVTQNU (KIWTG% %LQGLQJDQGLQKLELWLRQRI,J$WR8. Relative Cell Number. 6Q GZENWFG VJCV VJG QDUGTXGF FKHHGTGPEGU YGTG ECWUGF D[ VJG EGNNVTCPUHGEVCPV UKOKNCTGZRGTKOGPVUYGTGRGTHQTOGFYKVJEGNNUYJKEJPCVWTCNN[GZRTGUU%&(QT VJKU RWTRQUG VJG OQPQE[VKE NKMG EGNN NKPG 7 YCU WUGF aIgA2 %QPVTQN GZRGTKOGPVU UJQYGF C UKIPKHKECPV GZRTGUUKQP QH %& QP 7 CPF DKPFKPI QH RWTKHKGF +I# YJKEJ YCU EQORNGVGN[ KPJKDKVGF D[ /; FCVC PQV UJQYP 9JGP RWTKHKGF HTCEVKQPU QH OQPQOGTKE +I# HTQO RCVKGPVU CPF 100 101 102 103 104 EQPVTQNU YCU VGUVGF HQT DKPFKPI YG CNUQ HQWPF C aIgA2 + UKIPKHKECPVN[TGFWEGFDKPFKPIQHO+I#HTQO+I#0RCVKGPVU mIgA (IgAN). FCVCPQVUJQYP 100 101 102 103 104. aIgA2 + mIgA (Control). 100 101 102 103 104. Log Fluorescence Intensity. Figure 6. Inhibition of aIgA2 binding to U937 cells. Inhibition of heat aggregated IgA2 (50 µg/ml) binding to U937 by preincubation with mIgA (50 µg/ml) of controls (n=3; 34.23±4.43%), patients with IgAN (n=3; 11.68±2.70*). From each group (n =3) one representative flow cytometry histogram is shown: aIgA2 (top), inhibition of aIgA2 binding by mIgA from patients with IgAN (middle) and as above with mIgA from controls (bottom). Filled histograms represent the conjugate controls, open histograms represent the binding of aIgA2 to U937 cells. Values in percentage aIgA2 binding inhibition ± SEM (*:P < 0.05).. 6JGTGFWEGFDKPFKPIQHO+I#VQ%&OKIJVNGCFVQCTGFWEGFKPVGTHGTGPEGYKVJ R+I#DKPFKPICPFVJGTGHQTGHCXQWTR+I#FGRQUKVKQPXKC%&KPVGTCEVKQP6QCUUGUU VJGECRCEKV[QHO+I#HTQORCVKGPVUYKVJ+I#0CPFEQPVTQNUVQKPVGTHGTGYKVJDKPF KPI QH R+I# 7 EGNNU YGTG RTGKPEWDCVGF YKVJ RWTKHKGF HTCEVKQPU QH O+I# CPF URGEKHKEDKPFKPIQHC+I#YCUVGUVGF+UQNCVGFO+I#HTQOEQPVTQNU P KPJKDKVGF C+I# DKPFKPI YKVJ v YJGTGCU O+I# KUQNCVGF HTQO RCVKGPVU YKVJ +I#0 P UJQYGF C UKIPKHKECPVN[ TGFWEGF KPJKDKVKQP QH v. 2 (KIWTG . ',6&866,21 +PVJKUUVWF[YGCUUGUUGFVJGDKPFKPIQH+I#QHFKHHGTGPVUK\GUVQ++#EGNNUVTCPU HGEVGF YKVJ JWOCP %& 9G WUGF GKVJGT RWTKHKGF +I# QT +I# FKTGEVN[ HTQO RC VKGPVCPFEQPVTQNUGTCVQCUUGUUDKPFKPIVQ%&%QORCTKUQPQHDKPFKPIDGVYGGP. 90.

(20) Reduced binding of mIgA from IgAN patients to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v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α 6JG DKPFKPI QH +I# VQ OGUCPIKCN EGNNU KU PQV UQNGN[ FG RGPFGPV QP VJG RTGUGPEG QH %& (QT KPUVCPEG +I# QT +I#EQPVCKPKPI KOOWPG EQORNGZGU ECP DG DQWPF XKC VJGKT OCPPQUG TGUKFWGU CPF VCTIGVGF HQT GPFQE[VQUKU XKCVJGOCPPQUGTGEGRVQT 'ZRTGUUKQPQHVJGOCPPQUGTGEGRVQTQPOGUCPIKCN EGNNU ECP DG WRTGIWNCVGF KP XKVTQ D[ 60(α CPF +.α 6JGTGHQTG KV KU OQUV NKMGN[VJCVOWNVKRNGTGEGRVQTUOKIJVDGTGURQPUKDNGHQTDKPFKPIQH+I#KPVJGMKFPG[. 91.

(21) Chapter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α CPF %α TGIKQP &GIN[EQU[NCVKQP QH VJG 0NKPMGF ECTDQJ[FTCVG TGUKFWGU KP VJKU %α CPF %α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

(22) Reduced binding of mIgA from IgAN patients to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

(23) Chapter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