Document GKbn2KJJBd4Ebevm8ymOx90eV

FX zo z. i T i :* j^-: e^surfl.issa.voi.7i.November. N atural killer cells in renal allograft rejection >; .1 * >t* V: 7 i` ii G. Cooksey*. R. A. R o b in sf and R. W . Blarney* *Department o f Surgery. City Hospital, Nottingham and ^Cancer Research Campaign Laboratory, University o f Nottingham. UK Correspondence to: Mr G. Cooksey, City Hospital. Hucknall Road. Nottingham HG5 1PB, UK Monoclonal antibody Anti Leu 7 has been used to enumerate peripheral blood N K cells in 20 renal aUoqrafi recipients. Blood was taken preoperalively and three times per week jo r the first month after irons, plantation; separated mononuclear cells were stained with Anti Leu 7 ihni analysed on a jluorescence activated cell sorter. Results were related to acute rejection episodes diagnosed clinically and confirmed by Trucut biopsy o f grafts. A reduction in the number o f Leu 7 positive celU occurred postoperativeiy. Nineteen out o f 20 patients showed a rise itj number of circulating N K cells during acute rejection episodes. Keywords: Natural killer ceils, renal allograft, acute rejection Natural killer cells arc mononuclear leucocytes which without prior stimulation kill several types of tumour derived or virus infected cultured target cells in vitro. They are otherwise negatively defined as non-T. non-B. non-adherent and nonphagocytic cells. They incompletely express T lymphocyte and myelomonocytic surface phenotypes1. Morphologically natural killer (NK) cells are a hetero geneous group of cells, a large fraction of which are large granular lymphocytes3. These arc distinguishable from T lymphocytes by their larger rim of cytoplasm which is stippled with azurophilic granules. Large granular lymphocytes con stitute between 5 and 15 per cent of normal human peripheral blood mononuclear cells. NK cells appear to play a part in allograft rejection. In bone marrow transplantation NK cells are probably major efTector cells in natural, radiation resistant rejection3. Increased NK activity has also been associated with clinical graft versus host disease"1. In acute rejection of solid organ grafts the role of NK cells is less clear. In murine models up to 10 per cent of mononuclear cells infiltrating rejecting renal allografts are NK cells with high cytolytic activity. Ncmlander5 has reported that infiltration of rat kidney allografts by NK cells occurs early in the rejection process, before that by cytotoxic T lymphocytes. In man NK activity is usually measured with a cytotoxicity assay using K562 cell line derived from the pleural effusion of a patient with chronic myeloid leukaemia. Using this assay Guillou et a ib have reported an increase in NK activity to be associated with episodes of acute rejection in 12 patients. These authors did. however, suggest that the considerable dav-to-dav variation in the susceptibility of target cells to Ivsis makes serial estimations of this kind difficult in iniornrfi Two monoclonal antibodies Anti Leu 77 and Anti Leu I I 8 are now available which have been developed to bind to surface antigens specific to NK cells. Anti Leu 7 binds to large granular lymphocytes and Anti Leu 11 to the majority of cells exhibiting natural killer activity. The aim o f this study was to enum erate N K cells in the peripheral blood of hum an renal allograft recipients using Anti L ei 7 and to relate these results to episodes of acute rejection. Ten millilitres of venous blood was taken pre-opera lively and then three times each week for the first month after transplantation. Mononuclear cells were separated from fresh heparinized Mood using a Ficoll hypaque density gradient (technique modified from Normals Ur- :nics 18 16 3 10 <c 8 6 : : M aterials and methods Twenty patients who received cadareric renal aHoc rafts in the Notiinuhain unit during IViO have been studied. l*rc-operaiively all patients received at least three blood transfusions. The minimum tissue typing match was I Ur identity. Maintenance immunosuppression consisted of 20 me prednisolone from the day of transplantation plus azathioprine 2 mg. kg daily. The treatment of re jection episodes consisted of a 12 day reducing course of oral prednisolone starting at 2(X) mg per dav reduced hv Umg cverv 1 davs 2- 0 ------------------------------------------------------------ ------ figure I ,VK.TihiT of Leu 7 poumc x fir1ml hlotij i/| _"i<* - .jiheforr trimypluituitiun cornpjr,;! h*i;/i _'ir uvr tun! in' ountlu.l -- 1 N a tu ra l k iller cells in renal a llo g ra ft rejectio n : G. Cooksev et al. Boyum'>l. Between 2-5 and 5 x 10* washed ceils were then labelled with Anti Leu 7 using an indirect fluorescent technique. The percentage of lymphocytes labelled with Anti Leu 7 was determined using a fluores cence activated cell sorter utilizing forward angle and VO' scatter gates to confine the analysis to lymphocytes (FACS IV. Bccton Dickinson). The total number of cells per millilitre of blood was calculated from this percentage, a white blood cell and a differential count from the same blood sample. Results have been related to episodes of acute rejection which were initially diagnosed on the conventional clinical and biochemical criteria of: graft swelling and tenderness: fluid retention: decreased urine output: hypertension: fever in the absence of infection and elevation of serum creatinine. In all cases a Trucut needle biopsy was performed, before (he start of treatment for acute rejection, for histological confirmation of the diagnosis. Technical and infective causes of graft failure were excluded by routine isotope scanning of aIlog rafts, urine and blood culture and viral serology. All rejection episodes documented received treatment, as outlined above, which on review we feel was justified in each case. fijurr 2 }*umber o f Leu 7 positive cells x iff4 it htood betore unJ oiler transplantation. Student's I test, t = 7-2'65: P < tl tto! Results The number of Leu 7 positive cells per millililre of blood before transplantation in our 20 patients and in 20 age and sex matched normal volunteers is shown in Figure /. There was no significant difference between these two croups. Students t = 0-543; P> 0-5. In the first 3 days after transplantation there was a reduction in the number of Leu 7 positive cells in all patients as shown in Figure 2. Student's paired r =7-23: P <0-001. Figure 5 represents the number of Leu 7 positive cells and creatinine levels of a typical patient, who received treatment for rejection on the tenth postoperative day. The previous day a rise in Leu 7 positive cells greater than two standard deviations above the mean of three postoperative values occurred along with a modest rise in serum creatinine level. In the 43 h before the start of treatment for rejection we have observed a rise in both the percentage of lymphocytes positive for Anti Leu 7 and in the actual number of such cells per millilitre of peripheral blood in 19 out of 20 patients as compared with previous samples Isec Figure 4i. Student's paired t = 3-768; 001>P>0(X)1. V . Days Hjvr J .Vumber ol Lett 7 positive celts tenntumous lute) and serum ereaimine levels \hroLen fuie) id one patient J urina the lirst three wivLv alter Natural killer c ells in renal alloq raft rejection: G. C ook sey et al. In 17 patients there were ihrccor more postoperative but prerejection values from which to derive a mean and standard deviation. In 12 eases the increase in Leu 7 positive cells at the onset of rejection was greater than (wo standard deviations above the mean, in four cases there was a rise which was less than two standard deviations and in one patient numbers fell. Following the start of treatment for rejection both p^. centage and actual numbers of Leu 7 positive cells promptly fc|| 0 pre-rejection levels. This fall was observed n tv in 1election episodes which were successfully treated and those three patient* where treatment was unsuccessful and graft failure ensued. Four patients had only small rises in numbers of Leu 7 positive cells at the onset of acute rejection episodes. These patients ail had low cell numbers in the pre- and postoperative samples when compared with the other patients. It is of interest that the one patient whose cell numbers fel[31 the onset of acute rejection (and before the start of treat menu was one of the three patients to lose graft function during the period of study. One patient also investigated during the period of study w*JS not included in the above analysis as no acute rejection has occurred in over 1 year of follow-up. Figures illustrates that the number of circulating Leu 7 positive cells remained at the posttransplant level for the whole 3 weeks in this patient. Figure -4 Slumber o f Leu 7 posture celts x Itr 1/ blond hetbre and at the onset o f acute rejection episodes. Student's paired t feji. t = S-TfiX: 0 0 J> P>iH)(j/ Discussion Monoclonal antibodies in conjunction with FACS analysis arc rapid and convenient probes with which to investigate natural killer cells in a variety of situations. The reproducibility i.; (his method makes it most suitable for the examination 01 .-rial samples as in this study (whereas the variability of the .'.362 cytotoxicity assay mentioned previously makes results 2 ,,ult to interpret in this situation). It is acknowledged that Anti Leu 7 docs not i Aa\ ;.]| circulating mononuclear ceils with NK activity and thaioxerlip with cells of suppressor phenotype does exist. Houevtr in a previous report of Helper and suppressor cells in ti ts stmc group of patients we have found no increase in Leu 2a suppressor cells to occur at the time of rejection10 whid* cowlhi account for our findings. There are several factors which may explain the oh-ewjcif .ill in number of Leu 7 positive cells after transplantation cal stress is known to alter immunc-compctencc; in Ivmphocvte responses to mitogenic stimuli a re well kn. ^rvta-fill after a variety of surgical procedures11. In addilu-A steroids are known to depress NK activity. Lcnr.. .i Figure 5 S umber ttt t.eu 7piistlwe Cells x ItF'uit hiood and sennit cretinithie lefeh nt *ir nitlent diunm ttie ltr\l three t*eeksulter triiu^pliinhitU' t .1 N a tu ra l k ille r cells in renal a llo g ra ft rejectio n : G . Cooksey et al. per. i (personal communication) have observed a fall in number of i' fell fjfculiiting Leu 7 positive cells to follow uncomplicated surgery 'll'n ,ych as cholecystectomy and femoropopliical bypass. However, cuu n these cases numbers returned to pre-operative levels within 7 days. tlV'v >11\ In studies of this kind the definition of acute rejection is a difficult problem and a potential pitfall. The histological features pfallografts seen in Trucut needle biopsies is not a gold standard II :u dii i tJli- *hch can be used alone. In this study the diagnosis of each acute rejection episode was reviewed by an experienced clinician who assessed all the clinical and histological findings together. In the majority of cases presented, acute rejection episodes were clear U.i* I:.;. cot events. This is due to the rather aggressive nature of rejection episodes seen in the first month after transplantation where low doses of prednisolone are used for maintenance immunosuppression. ip*:. In this study we have found a significant increase in circulating NK cells, as defined by Ami Leu 7. to occur in renal iJIograft recipients at the onset ofacute rejection episodes. While the role of NK cells in renal allograft rejection remains unresolved, our findings add to the increasing body of evidence which suggests that these cells are involved in rejection. The ! .*1 question remains as to whether this rise in NK cells represents the activation of an effector pathway actively involved in the rejection process or whether it is a non-specific finding reflecting ictivation of the immune-system. Recent d ata,: which has ihown NK activity to be augmented by Intcr-leukin 2 points to a regulatory role for NK cells in the immune network. Thus studies of the mechanism of acute rejection now need to consider the involvement of NK cells in this process. The conclusion of this study is that the number of circulating natural killer cells as defined by the monoclonal antibody Anti Leu 7 increases in the majority of renal allograft recipients undergoing acute rejection. Acknowledgements References 1. Orta Ido JR. Sharrow SO. Timoncn T. Hcrbcrman RB, Deter mination of surface antigens on highly purified human NK Cells by (low cytometry with monoclonal antibodies. J Immunol (9X1 ; 127: NO I-9. 2. Sakela E.Timoncn T. Annamari R. Hayry P. Morphological and functional characterization of isolated dTcctor cells responsible for human Natural Killer activity to fetal fibroblasts and activated cell line targets. Immim Ret' 1979; 44: 71-123. 3. Kicsslinc R, Hochman PS. Haller O. Shearer G.M. Wigzcl H. Evidence for a simuhr or common mechanism for natural killer cell aeuvttv and resistance to haemopoictic grafts. EurJ Immunol 1977: 7: 6*55-63. 4. Lopez C. Kirkpatrick D. Sorcll M. O'Reilly RV. Ching C. Association between pre-transplant natural kill and graft versus host disease after stem cell transplantation. Ltincei 1979: ii; 1103 6. 5. Nemlander A. Sakscla E. Hayry P. Arc 'natural killer' cells involved in allogralt rejection? EurJ Immunol 19S3: 13: J4X-50. 6. Guillou PJ. Hcgarty J. Ramsdcn C. Davison AM. Will EJ. Giles GR. Chances in human natural killer activity early and late after renal transplantation using conventional immunosuppression. Trantpluntutum 19X2:33:414-21. f 7J Abo T. Balch CM. A differentiation antigen of human NK and K cells identified hy monoclonal antibody lMNK-1). J Immunol 19X1:127:1024-9. Qk/ Lanier LL. Englcman EG.Gatcnby P. Babcock GF. Warner NL. Herzenberg LA. Correlation of functional properties of human lymphoid subsets and surface marker phenoiypcs using multiparameter analysis and flow cytometry. Immun Ree 19S3: 74: 147. 9. Boyum A. Separation ofleucocytcs from hlood and bone marrow, Setind J Clin Lub Inreit 19Mt; 21: Suppl 97. 10. Cooksey G. Reeve RS. Paterson AD, Colton RE. Blarney RW. Helper and suppressor T lymphocytes in renal allograft rejection. Br J Surti 19X4; 71: 3X6. " 11. Park SK. Brody JI. Wallace HA. Blakemorc NS. Immuno suppressive effect of surgery. Lamvt 1971 : i: 53-5. 12. Suzuki R. Manda K. Itoh K. Kumagai K. Natural killer cells as a responder to Interleukin 2. J Immunol I9S3: 130: 9X1-7. We thank John Lawry and Owen Roberts for their considerable help "with FACS analysis of bur specimens. ; R. A. Robins is supported by the Cancer Research Campaign. Paper accepted 15 August 19X4