natural killer ( nk ) cells are large granular lymphocytes that have the ability to kill cells without prior sensitisation and therefore play an important role in host defence . nk cell - mediated target cell killing is mainly implemented by two pathways , namely the perforin / granzyme pathway and the fas ligand ( fasl ) pathway [ 2 - 5 ] . in the latter pathway , fasl on effector cells binds fas present on the target cells which results in oligomerization of fas and activation of caspase 8 . perforin and granzymes , of which granzyme b is the most potent one , reside in the granules of nk cells and are released by exocytosis after conjugation between the effector and target cell . inside the cytoplasm of the target cell , granzyme b activates caspase 3 directly   or indirectly , via a mitochondrion - dependent pathway . nk cells display two types of surface receptors : ( i ) activation receptors , such as the cd161 molecule that recognises structures on target cells and triggers nk cells to kill ; ( ii ) inhibitory receptors , such as ly-49 molecules , that recognise target cell mhc class i molecules and inhibit killing by nk cells . when mhc class i molecules are absent or expressed in reduced amounts , the nk cells proceed with their attack . a recent study shows that h-2dmhc class i molecules on target cells partially inhibit granzyme a release from mouse ly-49ank cells . however , it is unclear whether such partial inhibition of granzyme a release is sufficient to protect target cells . moreover , the assay used in the past to detect cytotoxicity by cytolysis is the release of cr from loaded target cells . a recent study questioned the relevance of the cr release assay compared to what occurs in vivo , whereas the dna fragmentation assay to measure apoptosis coincided with in vivo results . therefore , it is needed to explore whether the protective role of mhc class i is also operative in apoptosis induced by nk cells . compared with nk cells from spleen and peripheral blood , hepatic nk cells , also called pit cells , are much more cytotoxic . strategically located in the liver sinusoids , they constitute a first line of cellular defence against invading cancer cells , like colon carcinoma cells [ 15,17 - 20 ] . in this study , using freshly isolated hepatic nk cells and cc531s , a syngeneic fas ligand - resistant colon carcinoma cell line , we ( i ) demonstrated that mhc class i protects colon carcinoma cells from hepatic nk cell - mediated killing ; and ( ii ) showed the involvement of the perforin / granzyme pathway in the mechanism of mhc class i protection . protection of target cells from nk cell lysis by expression of mhc class i molecules has been demonstrated in different experimental systems in human , mouse   and rat . in rat , several mhc class i genes have been identified , i.e. , rt1.a , rt1.c and rt1.e . it has been shown that transfection of rt1.a and rt1.c protects target cells from lysis by nk cells . however , other data indicate that rt1.a molecules inhibit nk cells , whereas rt1.c region molecules activate natural killing . masking of rt1.a , rt1.c , or both alleles on target cells with allele - specific mabs , has no effect on lysis by nk cells . in view of these facts , mab ox18 was chosen to investigate the mechanism of mhc class i protection of cc531s target cells from hepatic nk cell - mediated killing . it has been found that ( i ) mab ox18 binds total rat mhc class i , ( ii ) masking of mhc class i molecules on target cells by mab ox18 or f(ab')2 fragments of ox18 enhances the syngeneic target cell cytolysis by rat nk cells , and ( iii ) the enhanced nk cell - mediated target lysis by mab ox18 is not caused by antibody dependent cellular cytotoxicity ( adcc ) . the expression of mhc class i molecules on cc531s cells was examined by flow cytometry . in agreement with a previous study , the mab cc52 , used as a negative control during functional assays , was shown to bind to cc531s cells , as has also been shown previously   ( data not shown ) . when cc531s cells were preincubated with mab ox18 against mhc class i molecules , the hepatic nk cell - mediated cytolysis against cc531s cells was increased in comparison with the lysis of untreated or control mab treated tumour cells ( fig . similarly , the preincubation of cc531s cells with mab ox18 increased fragmented dna ( apoptosis ) in cc531s cells when coincubated with hepatic nk cells ( fig . cc531s cells showed the typical morphological characteristics of apoptosis such as nuclear fragmentation , chromatin condensation ( fig . when cc531s cells were pretreated with mab ox18 , the number of apoptotic cc531s cells increased ( figs . it has been reported that anti - mhc class i antibody alone can induce apoptosis in cancer cells . in order to address the question whether the enhanced apoptosis and cytolysis in cc531s cells was induced by the binding of mab ox18 after 3 , 24 or 48 hours of incubation , no apoptosis or cytolysis in cc531s cells was observed ( data not shown ) . this is the first time it is shown that , besides cytolysis , mhc class i molecules protect cancer cells from apoptosis induced by nk cells . this is relevant because it has been suggested that in vitro assays quantifying effector cell - mediated apoptosis , but not cytolysis , are in accordance with in vivo results . effect of anti - mhc i mab ox18 on hepatic nk cell - mediated cc531s cell cytolysis ( a , cr release ) and apoptosis ( b , dna fragmentation ) . ( a ) , cr - labeled cc531s cells were incubated at an e : t ratio of 10:1 with freshly isolated hepatic nk cells for 18 hours . ( b ) , [ h]-tdr labeled cc531s cells were incubated at an e : t ratio of 10:1 with hepatic nk cells for 3 hours . conab , control antibody . * p < 0.05 , * * p < 0.01 vs. the treatment with medium only ( lsd test ) . hepatic nk cell - induced apoptosis in cc531s cells as observed by fluorescence microscopy ( a , c , e ) and light microscopy ( b , d , f ) . cc531s cells were coincubated with hepatic nk cells at an e : t ratio of 10:1 for 3 hours . ( a , b ) , coincubation of cc531s cells with hepatic nk cells in medium . ( e , f ) , cc531s cells were pretreated with anti - mhc i mab ox18 and hepatic nk cells were pretreated with dci . ( g ) , the percentage of apoptotic cc531s cells was determined in preparations by counting at least 300 cells per sample . p < 0.05 , * * p < 0.01 vs. the corresponding control ( lsd test ) . to address the mechanism of protection of mhc class i on cc531s cells , we used several approaches to assess the granule exocytosis pathway : granzyme inhibition by dci , cachelation by egta and caspase inhibition by z - vad - fmk . when hepatic nk cells were preincubated with dci , a granzyme inhibitor in intact cells , hepatic nk cell - mediated cc531s cytolysis was largely inhibited ( fig . 3a ) and apoptosis was completely inhibited in the ox18-treated and untreated cc531s cells ( fig . 3b and figs . effect of dci and egta on anti - mhc i mab ox18 enhanced cytolysis ( a , cr release ) and apoptosis ( b , dna fragmentation ) of cc531s cells by hepatic nk cells . cc531s cells were pretreated with mab ox18 and hepatic nk cells were pretreated with dci . ( a ) , cytolysis was determined by a 18 hour cr - release assay . ( b ) , apoptosis was determined by a 3 hour quantitative dna fragmentation assay . the e : t ratio was 10:1 . * * p < 0.01 vs. the corresponding control ( lsd test ) . the perforin / granzyme pathway is ca - dependent , involving extracellular caat three distinct steps : ( i ) granule exocytosis ; ( ii ) binding of secreted perforin to the membrane of target cells ; and ( iii ) perforin polymerisation . when extracellular cawas chelated by egta during the co - incubation , the anti - mhc class i mab - enhanced cytolysis and apoptosis of cc531s cells by hepatic nk cells was completely inhibited ( figs . 2 , 3 ) . the results obtained by granzyme inhibition and cachelation strongly suggest the involvement of the perforin / granzyme pathway in the anti - mhc i mab ox18 enhanced apoptosis and cytolysis . in order to verify these results , we made use , in a separate series of experiments , of the pan - caspase inhibitor z - vad - fmk . it has been shown that in several apoptotic pathways , including the perforin / granzyme pathway and the fasl pathway , dna fragmentation is caspase dependent . on the other hand , cytolysis is also caspase dependent in the fasl pathway , but caspase independent in the perforin / granzyme pathway [ 31 - 33 ] . as a consequence , this indeed , when z - vad - fmk was present during the coincubation of cc531s cells with hepatic nk cells , the ox18 enhanced apoptosis was completely inhibited , while cytolysis was not inhibited at all ( fig . fragmentation of the nucleus and condensation of the chromatin were also inhibited ( data not shown ) . effect of the pan caspase inhibitor z - vad - fmk on anti - mhc i mab ox18 enhanced cytolysis ( a , cr release ) and apoptosis ( b , dna fragmentation ) . cc531s cells were incubated at an e : t ratio of 10:1 with hepatic nk cells for 18 h ( cr release ) or 3 h ( dna fragmentation ) . * * p < 0.01 vs. the corresponding control ( lsd test ) . mhc class i expression protects colon carcinoma cells from apoptosis and cytolysis induced by hepatic nk cells , by blocking the perforin / granzyme pathway . this mechanism of immune escape could possibly contribute to the incomplete killing by hepatic nk cells of arriving colon carcinoma cells in the liver sinusoids , resulting in the formation of liver metastases . hepatic nk cells were isolated and purified from 3- to 5-month old male wag / rij rats ( rt1 , a wistar - derived inbred strain , harlan , the netherlands ) according to the method described before . the purity of the isolated hepatic nk cells was at least 90% , as evaluated by light microscopy using may - giemsa staining cytospins and by flow cytometric analysis using mab 3.2.3 , which recognises cd161a molecules on the surface of rat nk cells . the viability of the recovered cells was more than 95% , as determined by trypan blue exclusion . the procedures used in this study were approved by the local ethical committee ( license no . cc531s , a dimethylhydrazine - induced colon carcinoma of wag / rij rats , was maintained in culture medium rpmi-1640 ( gibco , life technologies , gent , belgium ) , supplemented with 10% fetal calf serum ( eurobiochem , bierges , belgium ) , penicillin ( 100 u / ml ) , streptomycin ( 100 g / ml ) , and glutamin ( 0.2 mm ) ( gibco , life technologies , gent , belgium ) . 3,4-dichloroisocoumarin ( dci ) and ethylene glycol - bis(-aminoethyl ether)-n , n - tetraacetic acid ( egta ) , were purchased from icn ( asse - relegem , belgium ) . the monoclonal antibody ( mab ) ox18 ( anti - rat mhc class i , igg1 )   was purchased from ecacc ( porton down , salisbury , uk ) . mab cc52 ( igg1 )   was developed in the department of surgery and pathology , leiden university medical center , the netherlands . z - val - ala - asp(ome)-fluoromethylketone ( z - vad - fmk ) was obtained from bachem ( bubendorf , switzerland ) . the expression of mhc class i molecules on the cc531s cells was determined by one - colour flow cytometric analysis , as described previously . briefly , 0.5  10cells were incubated ( 30 minutes , 4c ) with the primary antibody ox18 . cells were then washed three times with cold phosphate - buffered saline ( pbs ) , containing 1% bovine serum albumin and 0.02% sodium azide . subsequently , cells were incubated with fitc - conjugated antimouse igg1 ( gilbertsville , pa ) . after incubation and washing , cells were fixed with 2% paraformaldehyde in pbs and analysed ( facsort ; becton dickinson , mountain view , ca , usa ) . [ methyl - h]thymidine ( [ h]-tdr ) labeled cc531s cells were preincubated with the mab ox18 ( final concentration was 10 g / ml ) for 15 minutes , at room temperature , and freshly isolated hepatic nk cells were preincubated with 50 m dci for 30 minutes . it was shown that mab cc52 binds to cc531s cells   and is used in this study as a negative control in the functional assays . egta ( final concentration was 5 mm ) and z - vad - fmk ( final concentration 80 m ) were present during coincubation . the hepatic nk cells ( 10cells in 100 l ) and cc531s cells ( 10cells in 100 l ) were placed in triplicate in 1.5 ml microcentrifuge tubes . after 3 hours coincubation and centrifugation , the incubation medium was removed from the tubes . the lysates were ultracentrifuged ( 10,000 g for 15 minutes at 4c ) to separate fragmented dna from intact dna . radioactivity ( cpm ) in the incubation medium , in the 10,000 g supernatant and in the 10,000 pellet was determined in a  counter ( beckman , fullerton , ca , usa ) . the percentage of fragmented dna was calculated using the following formula : in which : cpmfr = the radioactivity in the incubation medium plus the cpm in the 10,000 g supernatant ; cpmtotal = cpmfr + radioactivity in the 10,000 g pellet ; exp = experimental ( target cells with effector cells ) ; spont = spontaneous ( target cells and medium only ) . cc531s cells ( 10cells in 100 l ) , were coincubated with hepatic nk cells ( 10cells in 100 l ) in a flat - bottom 96-multiwell plate , for 3 hours at 37c . after coincubation , nuclei of the cells were stained with hoechst 33342 and propidium iodide , as described previously . preparations were studied with a leica dm irb / e inverted fluorescence microscope ( leica , heidelberg , germany ) with ultraviolet excitation , at 340 to 380 nm . the images were recorded and the number of apoptotic cc531s cells was determined by the characteristic morphological changes of the apoptotic nucleus , i.e. , condensation of chromatin and nuclear fragmentation . cc531s cells , not coincubated with hepatic nk cells , were used as a control . the fragmented nuclei were counted in at least 300 cells in each preparation and the percentage of apoptotic cc531s cells was calculated using the following formula : cytolysis was measured in a 18 hour cr - release assay using 96-multiwell plates , as described previously . briefly , cr - labeled cc531s cells and hepatic nk cells were treated with the mab or dci or egta or z - vad - fmk , as mentioned above . cc531s cells at a concentration of 10cells / well were coincubated with hepatic nk cells at an effector - to - target ( e : t ) ratio of 10:1 in a final volume of 200 l . after 18 hours coincubation , the supernatant of each well was aspirated and radioactivity was determined in a  counter . the cpm usually ranged from 1200 cpm ( spontaneous release ) to 5000 cpm ( maximal release ) . results were expressed as percentage of specific lysis according to the formula : in which : exp = experimental ( target cells with effector cells ) ; spont = spontaneous ( target cells and medium only ) ; max = maximal , obtained after the addition of sds ( 1% final concentration ) . statistical analysis was performed by one - way anova ( n = 3 in each group , unless otherwise indicated ) with post - hoc multiple comparison analysis made by lsd ( the least - significant difference ) test , using spss statistical package ( spss inc . , statistically significant difference between two groups was considered at the level of p < 0.05 . hepatic nk cells were isolated and purified from 3- to 5-month old male wag / rij rats ( rt1 , a wistar - derived inbred strain , harlan , the netherlands ) according to the method described before . the purity of the isolated hepatic nk cells was at least 90% , as evaluated by light microscopy using may - giemsa staining cytospins and by flow cytometric analysis using mab 3.2.3 , which recognises cd161a molecules on the surface of rat nk cells . the viability of the recovered cells was more than 95% , as determined by trypan blue exclusion . the procedures used in this study were approved by the local ethical committee ( license no . cc531s , a dimethylhydrazine - induced colon carcinoma of wag / rij rats , was maintained in culture medium rpmi-1640 ( gibco , life technologies , gent , belgium ) , supplemented with 10% fetal calf serum ( eurobiochem , bierges , belgium ) , penicillin ( 100 u / ml ) , streptomycin ( 100 g / ml ) , and glutamin ( 0.2 mm ) ( gibco , life technologies , gent , belgium ) . 3,4-dichloroisocoumarin ( dci ) and ethylene glycol - bis(-aminoethyl ether)-n , n - tetraacetic acid ( egta ) , were purchased from icn ( asse - relegem , belgium ) . the monoclonal antibody ( mab ) ox18 ( anti - rat mhc class i , igg1 )   was purchased from ecacc ( porton down , salisbury , uk ) . mab cc52 ( igg1 )   was developed in the department of surgery and pathology , leiden university medical center , the netherlands . z - val - ala - asp(ome)-fluoromethylketone ( z - vad - fmk ) was obtained from bachem ( bubendorf , switzerland ) . the expression of mhc class i molecules on the cc531s cells was determined by one - colour flow cytometric analysis , as described previously . briefly , 0.5  10cells were incubated ( 30 minutes , 4c ) with the primary antibody ox18 . cells were then washed three times with cold phosphate - buffered saline ( pbs ) , containing 1% bovine serum albumin and 0.02% sodium azide . subsequently , cells were incubated with fitc - conjugated antimouse igg1 ( gilbertsville , pa ) . after incubation and washing , cells were fixed with 2% paraformaldehyde in pbs and analysed ( facsort ; becton dickinson , mountain view , ca , usa ) . [ methyl - h]thymidine ( [ h]-tdr ) labeled cc531s cells were preincubated with the mab ox18 ( final concentration was 10 g / ml ) for 15 minutes , at room temperature , and freshly isolated hepatic nk cells were preincubated with 50 m dci for 30 minutes . it was shown that mab cc52 binds to cc531s cells   and is used in this study as a negative control in the functional assays . egta ( final concentration was 5 mm ) and z - vad - fmk ( final concentration 80 m ) were present during coincubation . the hepatic nk cells ( 10cells in 100 l ) and cc531s cells ( 10cells in 100 l ) were placed in triplicate in 1.5 ml microcentrifuge tubes . after 3 hours coincubation and centrifugation , the incubation medium was removed from the tubes . the lysates were ultracentrifuged ( 10,000 g for 15 minutes at 4c ) to separate fragmented dna from intact dna . radioactivity ( cpm ) in the incubation medium , in the 10,000 g supernatant and in the 10,000 pellet was determined in a  counter ( beckman , fullerton , ca , usa ) . the percentage of fragmented dna was calculated using the following formula : in which : cpmfr = the radioactivity in the incubation medium plus the cpm in the 10,000 g supernatant ; cpmtotal = cpmfr + radioactivity in the 10,000 g pellet ; exp = experimental ( target cells with effector cells ) ; spont = spontaneous ( target cells and medium only ) . cc531s cells ( 10cells in 100 l ) , were coincubated with hepatic nk cells ( 10cells in 100 l ) in a flat - bottom 96-multiwell plate , for 3 hours at 37c . after coincubation , nuclei of the cells were stained with hoechst 33342 and propidium iodide , as described previously . preparations were studied with a leica dm irb / e inverted fluorescence microscope ( leica , heidelberg , germany ) with ultraviolet excitation , at 340 to 380 nm . the images were recorded and the number of apoptotic cc531s cells was determined by the characteristic morphological changes of the apoptotic nucleus , i.e. , condensation of chromatin and nuclear fragmentation . cc531s cells , not coincubated with hepatic nk cells , were used as a control . the fragmented nuclei were counted in at least 300 cells in each preparation and the percentage of apoptotic cc531s cells was calculated using the following formula : cytolysis was measured in a 18 hour cr - release assay using 96-multiwell plates , as described previously . briefly , cr - labeled cc531s cells and hepatic nk cells were treated with the mab or dci or egta or z - vad - fmk , as mentioned above . cc531s cells at a concentration of 10cells / well were coincubated with hepatic nk cells at an effector - to - target ( e : t ) ratio of 10:1 in a final volume of 200 l . after 18 hours coincubation , the supernatant of each well was aspirated and radioactivity was determined in a  counter . the cpm usually ranged from 1200 cpm ( spontaneous release ) to 5000 cpm ( maximal release ) . results were expressed as percentage of specific lysis according to the formula : in which : exp = experimental ( target cells with effector cells ) ; spont = spontaneous ( target cells and medium only ) ; max = maximal , obtained after the addition of sds ( 1% final concentration ) . statistical analysis was performed by one - way anova ( n = 3 in each group , unless otherwise indicated ) with post - hoc multiple comparison analysis made by lsd ( the least - significant difference ) test , using spss statistical package ( spss inc . , chicago , il , usa ) . statistically significant difference between two groups was considered at the level of p < 0.05 . adcc , antibody dependent cellular cytotoxicity ; cpm , counts per minute ; dci , 3,4-dichloroisocoumarin ; egta , ethylene glycol - bis(-aminoethyl ether)-n , n - tetraacetic acid ; fasl , fas ligand ; mab , monoclonal antibody ; mhc , major histocompatibility complex ; nk , natural killer ; z - vad - fmk , z - val - ala - asp(ome)-fluoromethylketone . p.j.k.k . provided the mabs ox18 and cc52 , and contributed to the text of the manuscript . supported by grants g038000 and g000599 from the fund for scientific research  flanders and grants ozr230 and ozr492 from the research council of the free university of brussels . we thank carine seynaeve and marijke baekeland for their technical help , karen crits for her technical assistance in flow cytometry , chris derom for her photographic support and ronald de zanger for his assistance in statistics .