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1 47 (5) :542546, 2001 A cta Zoologica S inica 3 NK / 33 (, ) 51 Cr 3 H2TdR N K ( PBN K) N K N K92 ( PAEC) ( HUV EC), N K92 PBN K PAEC HUV EC ; rhtnf2 PAEC/ HUV EC, PBN K TNF2 ; rhifn2 N K92/ PBN K, PAEC HUV EC ; CD11a (L FA2 1) PBN K TNF2 PAEC, N K PAEC HUV EC PBN KN K92, B. M. ) Na 51 2 CrO 3 4 H2TdR ( Amer2, sham ) MACS CD56 ( Mil2, tenyi Biotec ) CD11a ( HI111) ( Ig G1) ( Pharmingen ) 112, Jaffe (1973),, PBS, 011 %,, M199, 15 %FBS, 50g/ ml ( hyper acute rejection, HAR), 100 / ml, ( delayed xenograft rejection, DXR),,, 410, N K Monocytes (Blakely et al., 1994) 113 NK, N K92 Immue Medicine, Inc., Klingemann ( 1994 ), CD3 - / CD56 +, N K RP2, N K N K M I1640, 15 %FBS, 300 U/ ml rhil22 N K N K ( PBN K), ( ) N K Ficoll,, rh TNF2(R &D Systems ) rhifn2( , (No ) 33 ECGF, 100 g/ ml, mol/ L,, 35, :, MACS, CD56 PBN K, FACS Calibur (B. D. ), N K (CD3 - / CD56 + ) 90 %, N K92 PBN K 100 U/ ml hifn2 24 h
2 5 :N K / NK Mackay (1993) N K92,, N K92 10 %FBS PBNKNK92 PAEC/ HUVEC rh TNF2, N K92 PBN K HUV EC TNF2 24 h, , TNF23ng/ ml,, H2TdR, 1186 ( P < 0105) N K92 51 Cr PBN K, 37 5 % CO ( P < 0102) (1) rh T2 1 h 1640 N K NF2(21520 ng/ ml), N K92 PAEC 0125 % N K92, 215 % Tri2, ton X2100 PBN K 2 ( P > 0105) PBN K PAEC 3 H2TdR 51 Cr, TNF2,TNF210 ng/ ml,, 1172 ( P < : N K92 % = ) (2) H2TdR DPM 3 H2TdR DPM 100 % 213 IFN2 NK92/ PBNK PBN K % = 51 Cr CPM 51 Cr CPM 100 % N K U/ ml rhifn2 24 h, HUV EC PAEC, ( P < 0105),1166 ( P < 0101) 2 mmol/ L ED TA 015 %HSA PBN K rhifn2 24 h, PBS CD11a HU2V EC PAEC, ( HI111) Ig G1 37 PBN K 2145 ( P < 0102) 3112 ( P < 30 min, 01002) (2 : a, b) LFA21 NK t2 2, H2TdR, 37 2 h ; Chuluyan N K92 PBN K PAEC (1993) PBN K,, HUV EC ( P < 0101, P < 0102, t2, 5 % RPM I1640, ) (1 : a, b) N K92 N K Na 51 2 CrO 4 37 PBN K 1 h,,, PAEC HUV EC, 1640 PBN K ( P < 0105) 115 PAEC/ HUV EC 2 % rhtnf2 PBNKNK92 rh TNF2(15ng/ ml) PBN K CD11a, 1a. 1b. 1 NK PAEC/ HUVEC Fig. 1 Adhesion of human NK cells to PAEC/ HUVEC 3 H2TdR N K92, n = 6, E/ T 3/ 1 (N K92, n = 61 N K92 was labeled with 3 H2TdR, E/ T ratio was 3/ 1) 51 Cr PBN K, n = 4, E/ T 10/ 1 ( PBN K, n = 4, PBN K was labeled with 51 Cr, E/ T ratio was 10/ 1) 4 ( The wells were arranged in quadruplicates)
3 NK92/ PBNKHUVEC Table 1 The adhesion of NK92/ PBNK to HUVEC rhtnf2(ng/ ml) N K92 ( %) (N K92 adhesion ratio) PBN K ( %) ( PBN K92 adhesion ratio) (Notes) : SD ; HUVEC rhtnf2(05 ng/ ml) 24 h (N K92 : n = 5 ; PBN K: n = 4 ; 3 ) [ The data in the table was expressed as mean SD. HUVEC was prestimulated with rhtnf2(05ng/ ml) for 24 h (N K92 : n = 5 ; PBN K: n = 4 ; In triplicates) ] 2 NK92/ PBNKPAEC Table 2 The adhesion of NK92/ PBNK to PAEC rhtnf2(ng/ ml) N K92 ( %) (N K92 adhesion ratio) PBN K ( %) ( PBN K92 adhesion ratio) (Notes) : SD ; PAEC rhtnf2(020 ng/ ml) 24 h (N K92 : n = 5 ; PBN K: n = 4 ; 3 ) [ The data in the table was mean SD. PAEC was prestimulated with rhtnf2(020 ng/ ml) for 24 h (N K92 : n = 5 ; PBN K: n = 4 ; In triplicates) ] 2 IFN2 NK92/ PBNK Fig. 2 The effect of IFN2on the adhesion of NK92/ PBNK 2a. N K92 2b. PBN K n = 3 3 ( In triplicates) HUV EC TNF2 HUV EC, N K, 2211 % ( P < 0105) 5719 % ( P < 01002) 3 : a, PBN K CD11a, PAEC TNF2 PAEC,, N K 3816 % ( P < 0105) 2510 % ( P < 0105) 3 : b h TNF2 HUV EC PAEC, PBN K 3 ( Malyguine et al., 1996 ) N K PAEC HUV EC,, h TNF2,
4 5 :N K / LFA21 PBNKHUVEC/ PAEC Fig. 3 The effect of LFA21 on the adhesion of PBNK to HUVEC/ PAEC 3a. HUVEC, TNF2: 215 ng/ ml, n = 3 3b. PAEC, TNF2: 10 ng/ ml, n = 3 3, 3 TNF2 ( n = 3) ( The first three lanes were controls, while the last three lanes represented the EC stimulated with TNF2 ) h TNF2 N K HUV EC PAEC (Batten et al., 1996) N K92, TNF2, CD11a PBN K N K92 N K, PAEC HUV EC, 2211 % 5719 %, h TNF2 PAEC PAEC, N K92 IFN2 T N K,,, N K,, L FA21 ICAM21 ; IFN2 N K, PAEC HUV EC, ICAM21,, N K, L FA21 (CD11a, CD18) PAEC HUV EC,, N K, ICAM21, CD11a 3816 % 2510 %, PAEC TNF2, HUV EC TNF2, ICAM21, L FA21 PBN K HUV EC PAEC L FA21,,! ( References) Batten, P., M. H. Yacovb and M. L. Rose 1996 Effect of human cytokines ( IFN2, TNF2, IL21, IL24) on porcine endothelial cells : induc2 tion of MHC and adhesion molecules and functional significance of these changes. Im m unology 87 : Blakely, M. L., W. J. Van Der Werf and M. C. Berndt 1994 Activation of intragraft endothelial xenograft rejection. Transplantation 58 : & mononuclear cells during discordant Chuluyan, H. E. and A. C. Issekutz 1993 VLA24 integrin can mediate CD11/ CD182independent transendothelial migration of human monocytes. J. Cli n. Invest. 192 : Gong, J. H., G. Maki and H. G. Klingemann 1994 Characterization of a human cell line (N K92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 8 : Jaffe, E. A., R. L. Nachman, C. G. Becker and R. Minick 1973 Culture of human endothelial cells derived from umbilical veins. J. Cli n. In2 vest. 52 :
5 Mackay, F., H. Loetscher and D. Stueber 1993 TNF2 2induced cell adhesion to human endothelial cells is under dominant control of one TNF re2 ceptor type TNFR55. J. Ex p Med. 177 : Malyguine, A. M., S. Saadi, J. L. Platt and J. R. Dawson 1996 Human natural killer cells induce mophologica changes in porcine endothelial cell monolayers. Transplantation 15 : ( Abstract) D IFFERENTIAL AD HESION OF HUMAN NK CELLS TO PORCINE/ HUMAN VESSEL ENDOTHEL IAL CELLS 3 FEN G Zhi2Min ZHAN G Xiao2Feng FEN G Mei2Fu 33 ( State Key L ab. of Biomembrane and Membrane Biotechnology, Instit ute of Zoology, The Chi nese Academy of Sciences, Beiji ng , Chi na) The accumulated evidences demonstrate that natural killer (N K) cells may play an important role in the im2 mune cell mediated xenograft rejection. The adherent recognition of xenograft by N K cells is t he first and most important step, which N K cells may perform in the exnograft rejection. Endothelium is the first natural barrier between exnograft and host immune cells. So the study on adhesion between N K cells and endothelial cells is very important for preventing t he exnograft rejection. Using 51 Cr labelling and 3 H2TdR uptake, we studied the adhesions of human peripheral blood N K cells ( PBN K) and human N K cell line N K92 to porcine aortic endothelial cells ( PAEC) and human umbilical vein endothelial cells ( HUV EC). The results indicated that the adhesion ratioes of N K92 and PBN K to PAEC were markedly higher than those to HUV EC. The adhesion ratioes of PBN K showed that the h TNF2 2dose2depen2 dent increase after PAEC/ HUV EC were pretreated with h TNF2. The increases in adhesion ratioes of N K92/ PBN K prestimulated with hifn2to PAEC were significantly higher than those to HUV EC. Blocking with an2 ti2cd11a mab could differentially inhibit the adhesion of PBN K to resting and TNF2stimulated2PAEC. The results suggested that N K cells play an important role in the cell mediated xenograft rejection, L FA21 have the adherent activities across species and proinflammatory cytokines rh TNF2and could also activate PACE and HU2 VA E cells and exacerbate t he exnograft rejection. Key words Human, Porcine, PAEC, HUV EC, PBN K, N K92, Adhesion 3 This work was supported by National Natural Science Foundation of China (No ) 33 Corresponding author
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