(1)Shedding of CD89

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(7) Chapter 3. +PETGCUKPI EQPEGPVTCVKQPU QH TGEQODKPCPV U%& TGUWNVGF KP C FQUGFGRGPFGPV UKIPCNKPVJKU'.+5#6JGFGVGEVKQPNKOKVQHVJKU'.+5#YCUTGRTQFWEKDN[HQWPFVQ DG`RION (KIWTG 0RQRF\WLFFHOOVUHOHDVHV&'XSRQDFWLYDWLRQ 9G KPXGUVKICVGF VJG TGNGCUG QH UQNWDNG %& KP UWRGTPCVCPVU QH CEVKXCVGF JWOCP EGNNU(KTUVVJGTGNGCUGQH%&YCUUVWFKGFKPVJGRTQOQPQE[VKEEGNNNKPG7C EGNN V[RG GZRTGUUKPI JKIJ NGXGNU QH %& 5VKOWNCVKQP QH 7 YKVJ 2/# CPF KQPQO[EKPEQPUKUVGPVN[KPFWEGFTGNGCUGQHCUQNWDNGHQTOQH%& (KIWTG# A. U937. B. PMN 100. 100 PMA/ionomycin. sCD16 sCD89. medium 10. (ng/ml). sCD89 (ng/ml). 10. 1. 1. 0,1. 0 0. 1. 10. Time (hours). 100. 0 0. 1. 10. 100. 1000. Time (minutes). Figure 2. Detection of sCD89 in supernatants of PMA/ionomycin-stimulated cells. A. U937 cells were cultured in medium alone (open triangles) or medium supplemented with PMA/ionomycin (closed triangles). At indicated time points supernatants were tested for sCD89. Results are expressed as the mean ± SEM of 3 independent experiments. B. Freshly isolated PMN were cultured in medium with PMA/ionomycin. At the indicated time points supernatants were harvested and tested for sCD89 (closed triangles) and sCD16 (open circles). Results are expressed as a mean ± SEM of 3 independent experiments, using cells from 3 different donors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

(8) Shedding of CD89. 5HJXODWLRQRI&'VXUIDFHH[SUHVVLRQ. Figure 3. FACS analysis of CD89 surface expression. U937 cells were cultured in medium alone or in medium supplemented with PMA/ionomycin, LPS or heat aggregated IgA. After a 36-hour culture, CD89 surface expression was assessed with CD89 mAb 2D11 (1/1,000) followed by PE-conjugated goat antimouse-IgG1. Hatched histograms represent conjugate controls; open histograms represent CD89 expression. Out of 3 independent experiments one histogram is shown per condition. The dotted line represents the median fluorescence for CD89 expression of cells cultured in medium alone.. Relative cell number. 5KPEG OQFWNCVKQP QH UWTHCEG GZRTGUUKQP OKIJV EQPVTKDWVG VQ VJG TGNGCUG QH %& YG KPXGUVKICVGF VJG GHHGEV QH 2/#KQPQO[EKP QP %& OGODTCPG GZRTGUUKQP CU FGVGEVGFYKVJOQPQENQPCNCPVKDQFKGU(#%5CPCN[UKUUJQYGFVJCV2/#KQPQO[EKP KPFWEGF C HQNF TCPIG QH KPFGRGPFGPV GZRGTKOGPVU KPETGCUG KP Medium LPS %&UWTHCEGGZRTGUUKQP QP 7 EGNNU EQORCTGFYKVJEGNNUEWNVWTGFKPOGFKWO CNQPG (KIWTG . 100. 101. 102. 103. 104. 100. 101. PMA/ionomycin. 100. 101. 102. 103. 103. 104. 103. 104. aIgA. 104. 100. 101. 102. Log fluoresence intesity. A. B. 40. 20 anti-CD89 (2D11). sCD89 in ng/ml. U937. sCD89 in ng/ml. 102. 30. 20. 10. control (IgG1). 15. 10. 5. 0. 0 Med.. P/I. LPS. aIgA2. pIgA1. mIgA. 0,1. 1. 10. IgG1 in µg/ml. Figure 4. CD89-specific crosslinking triggers sCD89 release from U937. A. U937 cells were cultured in medium alone or in medium supplemented with PMA/ionomycin, LPS or IgA preparations consisting of heat aggregated IgA2 (aIgA2) from a patient with an IgA2 myeloma, polymeric IgA1 (pIgA1) from a patient with an IgA1 myeloma or monomeric serum IgA (mIgA) as indicated. After 36 hours of culture, supernatants were collected and tested for sCD89. Results are expressed as the mean ± SEM of 3 independent experiments. B. U937 cells were cultured in medium supplemented with different concentrations of the anti-CD89 mAb 2D11 (closed triangles) or an isotype matched control (open triangles). After 36 hours supernatants were collected and tested for sCD89. Results are expressed as the mean ± SEM of 3 independent experiments.. 55.

(9) Chapter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α4+ETQUUNKPMKPI WUKPI FKHHGTGPV EQPEGPVTCVKQPU QH VJG CPVK%& O#D & CPF HQWPF C FQUGFGRGPFGPV KPFWEVKQPQHU%&TGNGCUG (KIWTG$ #PKUQV[RGOCVEJGFEQPVTQNCPVKDQF[FKF PQVKPFWEGVJGTGNGCUGQH%&YJGTGCUETQUUNKPMKPI&WUKPIIQCVCPVKOQWUG +I)CPVKDQFKGUKPETGCUGF%&UJGFFKPI A. C. 1.5. 30 THP-1. sCD89 in ng/ml. sCD89 in ng/ml. Monocytes. 1.0. 0.5. 0. 20. 10. 0 Med.. P/I. LPS. aIgA2 anti-CD89 (2D11). Med.. P/I. LPS. aIgA2 anti-CD89 (2D11). B 30. sCD89 in ng/ml. MonoMac-6 20. 10. 0 Med.. 56. P/I. LPS. aIgA2 anti-CD89 (2D11). Figure 5. CD89 release from monocytic cell lines. CD89-expressing cells, peripheral blood monocytes (A), MonoMac-6 (B) and THP-1 (C), were cultured in medium alone or in medium supplemented with PMA/ionomycin, LPS, heat aggregated IgA2 or anti-CD89, as indicated. After 36 hours supernatants were collected and tested for sCD89. Detection limit is shown (100 pg/ml; thin dotted line). Results are expressed as the mean ± SEM of 3 independent experiments..

(10) Shedding of CD89. 5KOKNCT CEVKXCVKQP EQPFKVKQPU YGTG CRRNKGF VQ HTGUJN[ KUQNCVGF RGTKRJGTCN DNQQF OQPQE[VGU CPF VYQ QVJGT O[GNQKF EGNN NKPGU /QPQ/CE CPF 6*2 #EVKXCVKQP YKVJ 2/#KQPQO[EKP QT +I# UVKOWNCVGF CP KPETGCUGF UWTHCEG GZRTGUUKQP QP CNN VJTGGEGNNV[RGU FCVCPQVUJQYP +PCFFKVKQPDQVJ2/#KQPQO[EKPCPFC+I#CU YGNNCUCPVK%&CPVKDQFKGUKPFWEGFVJGTGNGCUGQHUQNWDNG%& (KIWTG 7KH)F5γFKDLQLVHVVHQWLDOIRU&'WULJJHUHGUHOHDVHRIV&' 6Q UVWF[ VJG OGEJCPKUO QH %&VTKIIGTGF TGNGCUG QH U%& KP OQTG FGVCKN YG WUGF OWTKPG ++# EGNNU VTCPUHGEVGF YKVJ JWOCP %& CNQPG QT YKVJ JWOCP %& KP EQODKPCVKQP YKVJ VJG (E4 γEJCKP UWDWPKV $QVJ VTCPUHGEVCPVU YGTG RTGXKQWUN[ UJQYP VQ JCXG C EQORCTCDNG %& GZRTGUUKQP CPF DQVJ EGNN NKPGU FKURNC[GFCUKOKNCT+I#DKPFKPI #EVKXCVKQPQH%&γEJCKPVTCPUHGEVGFEGNNU YKVJ KPETGCUKPI COQWPVU QH JGCV CIITGICVGF JWOCP +I# C+I# VTKIIGTGF C FQUG FGRGPFGPV TGNGCUG QH U%& 0Q U%& EQWNF DG FGVGEVGF KP UWRGTPCVCPVU QH C+I#UVKOWNCVGFEGNNUVTCPUHGEVGFYKVJ%&CNQPG (KIWTG# B. A γ/CD89. 6. sCD89 in ng/ml. sCD89 in ng/ml. 6. 4. 2. γ/CD89. 4. 2. CD89 0 Med.. 1. 10. 0. 100. aIgA in µg/ml CD89. sCD89 in ng/ml. 3. Figure 6. Release of CD89 from transfected cells is dependent on FcR γ-chain. A. CD89transfected cells were cultured in medium sup2 plemented with varying concentrations of aIgA2. After 36 hours, supernatants of stimulated IIA1.6 CD89/γ (closed squares) and IIA1.6 1 CD89 (open circles) cells were collected and tested for sCD89 protein. Results are expressed as the mean ± SEM of 3 independent 0 experiments. B. CD89-transfected cells were Medium P/I anti-CD89 anti-CD89 cultured in medium alone or medium with (2D11) (7D7) PMA/ionomycin, or anti-CD89 mAb’s 2D11 and 7D7. After 36 hours supernatants of the stimulated IIA1.6 CD89/γ (closed bars) and IIA1.6 CD89 (open bars) cells were collected and tested for sCD89 protein. Results are expressed as a mean ± SEM of 3 independent experiments.. 57.

(11) Chapter 3. 5KOKNCTN[ CPVK%& CPVKDQFKGU KPFWEGF TGNGCUG QH U%& QPN[ KP EGNNU EQ GZRTGUUKPI VJG γEJCKP (KIWTG $ 2/#KQPQO[EKP UVKOWNCVKQP NGF VQ TGNGCUG QH U%&KPDQVJEGNNV[RGU (KIWTG$ 6QKPXGUVKICVGYJGVJGTUKIPCNKPIXKCVJGγEJCKPKUKORQTVCPVHQTVJGTGNGCUGQH U%&VYQURGEKHKEKPJKDKVQTUYGTGWUGF#FFKVKQPQHGKVJGTCPKPJKDKVQTQHRTQVGKP MKPCUG % UVCWTQURQTKPG QT CP KPJKDKVQT QH RTQVGKP V[TQUKPG MKPCUGU IGPKUVGKP RTGXGPVGF VJG UJGFFKPI QH U%& HTQO VJG UWTHCEG QH C+I#UVKOWNCVGF ++# %&γEJCKPVTCPUHGEVCPVUQT7EGNNU (KIWTG . sCD89 in ng/ml. 15 IIA1.6CD89/γ. U937. aIgA. aIgA. 10. 5. 0 + Gen. + Stau.. + Gen. + Stau.. Figure 7. Shedding of sCD89 is prevented by genistein and staurosporine. Both the CD89/γ transfectants (left) and U937 cells (right) were cultured stimulated with aIgA2 in the absence or presence of an inhibitor of protein tyrosine kinases (gen. = genistein; 100µM) or an inhibitor of protein kinase C (stau. = staurosporine; 50 ng/ml). After 36 hours supernatants were collected and tested for sCD89 protein. Of three independent experiments, one repre-sentative example is shown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γ VTCPUHGEVGF ++# EGNNU NCPGU +P QTFGT VQ QDVCKP OQTG FGVCKNGF KPHQTOCVKQP QP VJG UVTWEVWTG QH U%& VJG RTQVGKP YCU RWTKHKGF HTQO UWRGTPCVCPV QH 2/#KQPQO[EKP CEVKXCVGF 7 EGNNU 5QNWDNG %& EQWNF DG RWTKHKGFWUKPICP+I#CHHKPKV[CDUQTDGPVYJKEJUJQYGFVJGPCVWTCNHQTOQHU%& VQ DG ECRCDNG QH DKPFKPI +I# 6JKU YCU EQPHKTOGF KP CP '.+5# U[UVGO YKVJ +I# EQCVGF VQ '.+5# YGNNU YJGTG C FQUGFGRGPFGPV DKPFKPI QH U%& VQ +I# YCU QDUGTXGF (KIWTG$ 9JGPVJGRWTKHKGFU%&YCUCPCN[\GFD[9GUVGTPDNQVVKPI 58.

(12) Shedding of CD89. YGQDUGTXGFCM&OQNGEWNG6TGCVOGPVYKVJ0IN[ECPCUGTGFWEGFVJG/9VQ M&(QTEQORCTKUQPVJGTGEQODKPCPVUQNWDNGHQTO M& YCUVTGCVGFKP VJGUCOGYC[CPFRTGFKEVCDN[UJQYGFCM&DCEMDQPGCHVGTTGOQXCNQHVJG0 NKPMGFIN[ECPU (KIWTG% . THP-1. IIA1.6 CD89/γ. Med P/I 6 7. Med aIgA2 kDa 8 9. U937. A. 1. 2. P/I* 5. Med P/I 3 4. 79. CD89. 57 37 sCD89. 29 Cells. Supernatants. B. C. 2,0. Recombinant sCD89. IgA coating BSA coating. OD415. 1,5. -. +. sCD89. -. +. kDa 57. 1,0. 37. 0,5. 29 0 0. 200. 400. 600. 800. 1000. 23. sCD89 in ng/ml. Figure 8. Immuno-chemical analysis of sCD89 protein. A. Cell lysates from U937 cells were blotted with normal rabbit and goat sera as a control (lane 1), or CD89 specific antisera (lane 2). Supernatants of U937 and THP-1 cells cultured in medium alone or stimulated for 36 hours with PMA/ionomycin (as indicated) were blotted with CD89 specific sera (resp. lane 3, 4 and 6, 7). As a specificity control (lane 5), rabbit and goat polyclonal anti-CD89 antibodies, were preincubated with 25 µg recombinant soluble CD89 and used for blotting of supernatant from PMA/ionomycin stimulated U937 (P/I*). Similar as for U937 and THP-1 supernatants of IIA1.6 CD89/γ cultured in medium alone or stimulated with aIgA2 were blotted (resp. lane 8, 9). B. Purified sCD89 was tested at the indicated concentrations for binding to IgA (coated 2 µg/ml, closed triangles) or BSA (coated 2 µg/ml, open squares)(as a specificity control). Results are expressed as the mean ± SEM of 3 independent experiments. C. Recombinant soluble CD89 and purified sCD89 were treated with N-Glycosidase F for removal of N-linked sugars. Deglycosylated or untreated samples, as indicated, were separated on 10 % SDS-PAGE and blotted specific for CD89.. 59.

(13) Chapter 3. ',6&866,21 +PVJGRTGUGPVUVWF[YGFGOQPUVTCVGVJCVWRQPCEVKXCVKQPO[GNQKFEGNNUECPTGNGCUG C UQNWDNG HQTO QH VJG (Eα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γ4+++ CPF %&(Eα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γ EJCKPYJKEJKUCNUQCUUQEKCVGF YKVJ (Eε4+ (Eγ4+++C%& CPF (Eγ4++C%& . (KIWTG # 5KIPCNKPI XKC VJKU UWDWPKV KPFWEGU 2-% CEVKXCVKQP CU YGNN CU V[TQUKPGRJQURJQT[NCVKQPQHVJG γ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γEJCKPUGGOUVQJCXGPQGHHGEVQPVJGCHHKPKV[HQT+I#6JGUG HKPFKPIU CTG FKHHGTGPV HTQO FCVC RWDNKUJGF HQT (Eγ4 CPF TGSWKTG HWTVJGT KPXGUVKICVKQP 6JG OQNGEWNCT YGKIJV QH M& QH %& TWNGU QWV VJG RQUUKDKNKV[ VJCV VJG RTQFWEVUOGCUWTGFKP'.+5#CTGTGNGCUGFOGODTCPGXGUKENGUEQPVCKPKPIHWNNNGPIVJ %&4GEGPVN[CVNGCUVFKHHGTGPVURNKEGXCTKCPVUQH%&JCXGDGGPKFGPVKHKGF 60.

(14) Shedding of CD89. +V UGGOU WPNKMGN[ VJCV VJG[ CTG TGURQPUKDNG HQT VJG U%& OQNGEWNG CU OQUV QH VJGO UJQYGF RCTVKCN QT EQORNGVG FGNGVKQPU QH '% QT '% YJKNG UVKNN EQPVCKPKPIVJGRTGFKEVGFVTCPUOGODTCPGTGIKQP(KPCNN[YGHQWPFVJCV++#EGNNU VTCPUHGEVGFYKVJHWNNNGPIVJ%&E&0#GZENWFKPICNVGTPCVKXGURNKEKPICNUQTGNGCUG CUKOKNCTM&OQNGEWNGWRQPCEVKXCVKQP (KIWTG#NCPG 6JGUGFCVCUWIIGUVC TQNG HQT RTQVGQN[VKE ENGCXCIG CU FGOQPUVTCVGF HQT XCTKQWU OQNGEWNGU KPENWFKPI E[VQMKPGU 60(α E[VQMKPG TGEGRVQTU CFJGUKQP OQNGEWNGU CPF (E TGEGRVQTU. 6JGENGCXCIGUKVGQH%&(Eγ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γ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

(15) Chapter 3. +P EQPENWUKQP KP VJG RTGUGPV UVWF[ YG JCXG UJQYP VJCV VJG O[GNQKF (Eα4+%&ECPDGTGNGCUGFCUCM&UQNWDNGOQNGEWNG6JGTGNGCUGQHU%& YJKEJECPDKPF+I#KUKPFWEGFWRQPCEVKXCVKQPQHO[GNQKFEGNNU6JKUOC[RTQXKFG C OQFG QH PHKPGVWPKPIQ GHHGEVQT HWPEVKQPU QH %& GZRTGUUKPI EGNNU +P TGEGPV [GCTUVJG%&OQNGEWNGJCUGXQNXGFCUCECPFKFCVGVCTIGVHQTDKURGEKHKECPVKDQF[ VJGTCR[ +V YKNN DG KORQTVCPV VQ WPTCXGN VJG OGEJCPKUOU QH %& UJGFFKPI PQV QPN[ VQ RQVGPVKCNN[ KORTQXG VJG GHHKECE[ QH VJGTCR[ DWV CNUQ VQ OQPKVQTKOOWPGCEVKXCVKQP. 62.

(16) Shedding of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

(17) Chapter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

(18) Shedding of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

(19) Chapter 3. 66.

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