since its introduction in   the late 1980s , laparoscopic cholecystectomy ( lc ) has been used as a  gold standard  for management of symptomatic gallstone disease , replacing almost completely the open cholecystectomy ( oc ) [ 13 ] . it is commonly followed by less pain , shorter hospitalization , and faster recovery owing to the reduced magnitude of the initial surgical insult and lower systemic immune impairment . however , positive co2 pneumoperitoneum , abdominal wall distention , and peritoneal acidosis may activate the additional factors that affect the outcome of operations , leading to both the potentiation of surgical trauma [ 4 , 5 ] , and the beneficial immune - modulating effect , linked with the augmentation of il-10 , attenuation of tnf production [ 68 ] , and diminishing effect on surgery - induced imbalance in pro-/anti - inflammatory cytokines [ 9 , 10 ] . cellular mechanisms underlying these effects are , however , very complex , since surgical manipulation or pneumoperitoneum and laparotomy may alter the expression of 116 and 398 splenic t - cell genes , respectively , that might be involved in signal transduction , chemotaxis , and cell cycle of lymphocytes .   in the present study , we tried to analyze the perioperative changes in cytotoxic t lymphocytes ( ctls ) and natural killer ( nk ) cells after lc and oc , since , particularly , these cells are involved in immune surveillance and have the crucial roles in the prevention or attenuation of disease through their ability to eliminate infected or transformed cells [ 1215 ] . their cytoplasm is rich in granules composed of complexes of proteoglycan in association with the pore - forming protein perforin ( p ) and a family of serine proteinases , known as granzymes that are released into the intercellular space on interaction of nk cells or ctl with a target cell . whether granzyme entry into the target cell cytoplasm occurs directly after perforin pore formation in the plasma membrane or after endosomolysis is still debated [ 1215 ] . according to the original model , p acts by forming a cell membrane pore through which granzymes pass into the target cell , where they cleave specific substrates that initiate dna fragmentation and caspase - dependent or caspase - independent apoptotic cell death . however , more recently , it was proposed that granzymes might be endocytosed independently of p and that p acts to release granzymes that are sequestered in endosomes into the cytosol of the target cell . in addition , there is a possibility that p triggers ca influx , which may activate rapid coendocytosis of granzymes and p into giant endosomes , followed by p - mediated release of granzymes to the cytosol [ 1215 ] .   in circulating nonactivated lymphocytes , cytotoxicity is restricted to nk cells , / tcr t cells , and a subpopulation of cd8 cells , which constitutively express perforin but its expression can be induced in many cd4 , mhc class ii restricted ctl , in cd8 ctl as well as in cd4cd25foxp3 t regulatory cells [ 1215 ] . moreover , the expression of p in t cells might be a useful marker for functionally activated cytotoxic cells in situ . trying to determine the early postoperative effects of classic and laparoscopic cholecystectomy on these events , in this study we analyzed the p content in the pbmnlc before and 24 hours after the operations . furthermore , in an attempt to link these events with the intensity of preoperative cholestasis and inflammation induced by gallstones , we correlated these data with the blood concentrations of acute phase proteins and markers of cholestasis , such as bilirubin ( bil ) , alkaline phosphatase ( ap ) , and hepatic enzymes . the data showed that perforin - dependent cytotoxic mechanisms are only moderately affected by different types of operations , but that the preoperative cholestasis and hepatic injury may significantly increase the number of peripheral blood cells containing the perforin . in a prospective , nonrandomized study that was approved by ethical committee of medical faculty , university of rijeka , we analyzed the immune functions of 23 patients submitted to conventional , open cholecystectomy ( oc ) and 21 patients undergoing laparoscopic cholecystectomy ( lc ) for symptomatic cholecystolithiasis . mean age for the conventional group was 61.8 years ( range 31 to 85 ) for 13 women and 10 men . for lc , mean age was 55.5 years ( range 28 to 82 ) ; this group consisted of 13 women and 8 men . laparoscopic cholecystectomy was carried out in a standardized technique using 4 troacars and a pneumoperitoneum of 14 mm hg . mean operative duration was 90 minutes ( range 74139 ) for oc and 120 minutes ( range 90150 ) , for lc without statistically significant difference , showing that the two groups were comparable for the demographic and surgery - related parameters ( table 1 ) . there were also no statistically significant differences in the standard hematological parameters , such as red and white cells counts , hematocrit , and platelets ( not shown ) but group of patients chosen for classic cholecystectomy had significantly greater preoperative cholestasis ( figure 1 ) , since laparoscopic cholecystectomy with insufflations of co2 was avoided in patients with chronic cholecystolithiasis . therefore , this group of patients had significantly higher levels of total bilirubin ( 57.6 versus 13.1 mol / l , p < .04 ) , ap ( 216.4 versus   64.0 iu / l ; p < .02 ) , ggt 273.03 versus 28.96 iu / l ; p < .004 ) , ast ( 56.3 versus 24.4 ; p < .05 ) , alt ( 95.42 versus 28.9 iu / l ; p < .009 ) , as well as the higher levels of crp ( 28.57 versus 4,84 mg / l ; p < the patients with acquired or inherited immunodeficiencies and those who developed postoperative complications were excluded from the study . blood samples were collected preoperatively and on postoperative day 1 and tested for hematological and immunological parameters . twenty healthy persons , consisting of laboratory personnel and blood volunteer donors ( 10 females and 10 males ) , corresponding by age and sexes to the examined patients groups were also studied . heparinized venous blood ( 10 ml ) was diluted and separated on lymphoprep ( nycomed pharmaas , oslo , norway ) for 20 minutes on 800 g. cells accumulating at the interface were washed twice in rpmi 1640 and resuspended at a final concentration of 1  10   peripheral blood lymphocytes per sample in fluorescent - activated cell sorting ( facs ) buffer . viability was assessed by trypan blue and was always around 98% . for staining of surface markers on human t and nk cells , the following phycoerythrin-(pe)- conjugated moab , obtained from becton dickinson ( mountain view ) were used : anti - cd3 , anti - cd4 , anti - cd8 , anti - cd16 , and anti - cd56 ) . for the detection of intracellular perforin a murine antihuman perforin moab g9 ( moab ; igg2b ) was purified from balb / c ascites . the method for the simultaneous detection of perforin ( intracellular antigen ) and cell surface antigens is described in detail elsewhere . briefly , peripheral blood mononuclear cells ( pbmnlc ) were aliquoted ( 10 per aliquot ) and washed in facs buffer , then fixed in 100 l pbs containing 4% paraformaldehyde , ph 7.4 , for 10 minutes at room temperature . after two washes in facs buffer , the cells were permeabilized with saponin buffer ( 0.1% saponin ) ( sigma , poole , dorset ) for 20 min at room temperature . murine monoclonal antibody reacting with human perforin ( p ) moab - g9 ( igg2b , purified from balb / c ascites ) was added to the suspension of cells in saponin , at final concentration of 810 g per sample and incubated for 30 minutes at room temperature . all further steps , secondary antibody , fitc - conjugated goat - antimouse ig ( becton dickinson , san jose , calif , usa ) , and rising procedures were performed in saponin buffer . cells surface antigens ( cd3 , cd4 , cd8 , cd16 , and cd56 ) were then stained in facs buffer at a final concentration of monoclonal antibody of 10 g / ml ( becton dickinson ) and 100 l were added to the pelleted cells . after 30-minutes incubation at + 4c , cells were washed with facs buffer and stained with avidin - phycoerythrin ( 20 g / ml , becton dickinson ) for 30 min . the standard preoperative workup for predicting choledocholithiasis in both group of patients included liver function tests and abdominal ultrasound . liver transaminases ( alt , ast ) , liver cholestasis tests ( gamma - glutamyl transpeptidase - ggt , and alkaline phosphatase - alp ) and liver clearance tests ( total and conjugated bilirubin - bil ) were determined by enzymatic calorimetric methods , using routine laboratory techniques with roche reagents in a roche - hitachi modular - p800 clinical chemistry module ( roche , mannhein , germany ) . statistical significance was calculated by two - tailed student 's t - test for paired samples , and by mann - whitney u - test for the evaluation of differences between the groups . test of correlation and linear regression analyses were made by statistical software statistics for windows , kernel release 5.5 ( statsoft , inc . linear regression equations were generated using a pearson - product moment correlation and analyzed for differences of covariance . data are reported as mean      sem or as median values , with 25th75th percentiles in parentheses . immune status of patients undergoing classic , open ( oc ) , or laparoscopic ( lc ) cholecystectomy was evaluated before and 24 hours after the operations . comparison of the data was then made between the pre- and postoperative values in the same group , between the preoperative values in the oc and lc groups as well as between the preoperative values in groups of patients elected for operations and the control group , consisting of healthy blood donors of corresponding age . analysis included standard determination of surface antigens on lymphatic cells and two color analysis , with simultaneous determination of surface markers and intracellular content of perforin . standard immunological tests revealed that preoperative numbers of t lymphocytes ( cd3 , cd4 , and cd8 ) and nk cells ( cd16and cd56 ) in both groups were in normal ranges for healthy , adult people . furthermore , no differences were found in preoperative values of both t ( figure 2(a ) ) and nk cells ( figure 2(b ) ) in blood of patients elected for oc and lc groups as well as in the postoperative values of all t lymphocytes and cd16 nk cells . classic cholecystectomy , however , slightly decreased the percentage of cd56nk cells ( 18.7% versus 15.4% ; p < .05 ) , as shown on figure 2(b ) . data were then analyzed by double labeling of cd surface marker and perforin and presented as a fraction of total peripheral blood lymphocytes containing perforin ( tp cells ) and as a percentage of double positive t   ( figure 3(a ) ) or nk cells ( figure 3(b ) ) . the significant postoperative difference was found only after lc , which decreased the fraction of cytotoxic cd8pcells from 13.5 to 10.2% ( p < .05 ) , as it is presented on figure 3(a ) and on representative histogram ( figure 3(c ) ) , showing perforin expression on the gated cd8 t cells , before and after lc .    comparison with healthy control revealed , however , that preoperative values of tp and cd3p cells in patients elected for oc were significantly higher than normal values ( 30.78% and 16.05% versus 27.3% and 10,68% , resp . ; p < .05 ) ( figure 3(a ) ) , as well as that all patients elected for operations had , preoperatively , lower fractions of perforin positive nk cells ( figure 3(b ) ) , since in oc group cd16p   cell fraction fell from 20.16% to 14.14% ( p < .05 ) and cd56p   cells from 20.74% to 13.63% ( p < .05 ) and in lc group to 10.71% ( p < .05 ) and to 11.64% ( p < .001 ) , respectively . increased expression of perforin in t cells ( cd3p and cd4p ) and lower expression of perforin in nk cells ( cd16p ) in preoperative blood of patients elected for classic cholecystectomy versus control subjects are presented also by the representative density plots ( figure 4(a ) and 4(b ) , resp . ) . the procedure of double labeling of peripheral blood lymphocytes by flow cytometry permitted also the measurement of mean fluorescence intensity ( mfi ) , which correlates with a number of perforin molecules present in the cells . by applying standard parameters for fsc , ssc , fl1 and fl2 , we thus , analyzed mfi for perforin in subsets of t lymphocytes   ( cd3p , cd4p , cd8p ) and nk cells ( cd16p and cd56p ) . as presented on figure 5(b ) and on representative histogram ( figure 5(c ) ) we noticed slight upregulation of perforin content only after lc in cd56pcells ( p < .05 ) . the data , however , clearly showed that lymphatic subpopulation ( cd3 , cd4 , cd16 and cd56 ) of patients undergoing the classic cholecystectomy contained , preoperatively , the higher quantities of perforin in their granules in comparison with patients selected for lc ( figures 5(a ) , 5(b ) , and 6 ) .   the findings of the marked changes in the proportions and mfi of perforin - expressing cells in preoperative blood suggested that these events might be related with the previously described greater cholestasis and inflammation in patients elected for classic cholecystectomy ( figure 1 ) . to test this possibility the concentrations of crp , hepatic enzymes and bilirubin in preoperative sera of all patients undergoing to the operations were , individually , in each person correlated with the percentage of perforin positive cells and with mfi for perforin in lymphocytes subpopulations found in preoperative samples of blood . it was found ( figure 7 ) that concentration of crp was in a very high positive correlation with percentage of total number of perforin positive cells ( r = 0.5218 ;   p < .002 ) , cd3p ( r = 0.49576 ;   p < .006 ) and the fraction of p cells among cd4 subset of cells ( r = 0.47551 ;   p < .006 ) as well as in the negative correlation with the fraction of pcells among cd16 cells ( r = 0.3752 ;   p < .041 ) .   the data ( figure 8) revealed also the presence of positive correlation between the concentration of ap in serum and proportion of cd4p cells ( r = 0.50240 ; p < .001 ) as well as the presence of negative correlation between ap and proportion of pcells among the cd8cells ( r = 0.3645 ;   p < .04 ) , and between the concentration of total bilirubin and proportion of p cells in cd8 cells ( r = 0.3655 ;   p < .034 ) .   however , although these data suggested that cholestasis and inflammation have induced a switch in immune response toward the cd4pcells , it was also found that higher concentrations of alt in sera increased the mfi of perforin in cd16 ( r = 0.409 ;   p < .016 ) and cd56cells ( r = 0.509 ;   p < .002 ) , suggesting that remaining nk cells , owing to the increased content of p probably obtained a higher cytotoxic activity ( figure 9 ) . trying to contribute to the elucidation of profound immunological dysfunctions induced by surgical trauma [ 111 ] and mechanisms that regulate the p expression in cytolytic cells [ 1215 ] , in this study we analyzed primarily the changes in perforin expressing peripheral blood lymphatic cells after oc and lc . the data showed that in early postoperative period , the type of operation minimally affected the p - related events , since only lc decreased the percentage of circulating cd8pcells ( figures   3(a ) , 3(c ) ) and increased the mfi of perforin in cd56cells ( figures   5(b ) , 5(c ) ) , suggesting that the pneumoperitoneum with insufflations of co2 had a stimulating effect on cytolytic activity of nk cells and contributed to distribution of ctl in the tissue . changes might be related with lower operative stress , extracellular acidification or selective activation of different macrophage subpopulation [ 111 ] but need further characterization , particularly , owing to our findings that immune outcome of cholecystectomy might be markedly influenced by the intensity of preoperative inflammation and cholestasis . in our study this , obviously , influenced the number of perforin expressing cells in both groups of patients since they , preoperatively , had significantly lower proportions of cd16p and cd56p cells ( figures 3(b ) and 4(b ) ) . similarly , augmented proportions of tp cells , cd3p cells and cd4p cells ( figures 3(a ) and 4(a ) ) were found in patients elected for oc , which preoperatively , had higher preoperative levels of crp , ap , b , ast and alt ( figure 1 ) , suggesting that preoperative inflammation and cholestasis affected the perforin - related events . the hypothesis was confirmed by linear regression analyses , made in all patients elected for the operations , which clearly showed that the concentration of crp in the serum was in high positive correlation with the percentage of tp cells , cd3p and cd4p cells and in negative correlation with the fraction of p cells among the cd16 nk cells ( figure 7 ) . moreover , an additional effect of cholestasis was confirmed by finding of positive correlation between ap in the serum and fraction of cd4p cells , and negative correlation of both   ap and bilirubin with fraction of p cells among cd8 cells ( figure 8) . taken together , the data imply that , in patients with gallstones , the inflammation and chronic cholelithiasis had particularly stimulated the generation of cytotoxic cd4p   cells , decreasing simultaneously the circulating levels of cd8p t cells and cd16pnk cells ( figures 3 , 4 , 6 , and   7 ) , probably owing to their greater homing into the insulted liver tissue . however , based on the fact that alt was in high positive correlation with mfi of perforin in cd16 and cd56cells ( figure 9 ) , we speculated also that hepatic injury might increased the content of perforin per cell in remaining circulating nk cells . the data are in high agreement with current knowledge about the influence of inflammation and injuries on cytotoxic cells of innate [ 18 , 19 ] and adaptive immunity [ 20 , 21 ] and point to the liver as an important organ for maintenance of self - tolerance and elimination of potentially dangerous cells that arose during the cholestasis and hepatic damages in patients with gallstones . the mechanisms are unclear , since the consequences of obstructive jaundice are complex and involve a number of immune effectors that contribute to a variety of immunological derangements [ 22 , 23 ] as well as to hepatic regeneration [ 24 , 25 ] and fibrosis   .   in this context it could be speculated that destruction of intrahepatic bile ducts or the appearance of aberrant cholangiocytes has initially stimulated the intrahepatic nk cells , leading to cholangiocyte killing and apoptosis by the granzyme / perforin pathway or by activation of death receptors belonging to the tumor necrosis factor ( tnf ) receptor superfamily and trail , as it was reported in the bile duct - ligated mice   and in patients with the primary biliary cirrhosis , where the activation of hepatic nk cells led to exacerbated damage on liver tissues [ 23 , 28 , 29 ] . noteworthy , higher cytotoxicity might be induced by activation of several classes of killer lectin - like receptors ( e.g. , nkg2d and natural cytotoxicity receptors ) , that are commonly expressed by human nk cells   as well as by tcr and tcrcd8 t lymphocytes and specifically interact with stress - inducible molecules , such as mica , micb , and ulbp , which might be displayed by    stressed  or  altered  self cells and by virus - infected cells . furthermore , since cholestatic jaundice is often followed by bacteriemia [ 22 , 23 ] , for our data showing the correlation of cd4p cells with the serum levels of crp and ap ( figures 7 and 8) might be relevant that in inflammatory bowel diseases with uncontrolled immune responses to bacterial flora were found cd4 ( + ) nkg2d ( + ) t - cell clones that were involved in perforin - mediated killing of the targets expressing mica . it should be , however , emphasized that cd4p cells t cells , activated in the liver , might belong also to cd4nkt cells or to cd4cd25 regulatory t cells , since these autoreactive t cells clones also participate in perforin - mediated killing . the former , which might be activated by glycolipid antigens , are characterized also by rapid activation and secretion of large amounts of numerous types of cytokines that in turn interact with a multitude of other cell types [ 3235 ] , while the latter are known   to participate in the control of autoantigen recognition and the development of autoreactive t cells clones , by mechanisms that include direct killing of cytotoxic cells through cell - to - cell contact , inhibition of cytokine production by cytotoxic cells and direct secretion of immunomodulatory cytokines , such as tgf- and interleukin-10 [ 3639 ] , acting together with other cd4 cells as crucial regulators of adaptive and innate immune responses . in conclusion , our data show that in early postoperative period only laparoscopic cholecystectomy moderately affected perforin - expressing cells in peripheral blood , decreasing the percentage of cd8p   cells and increasing the content of perforin in cd56   cells . however , pointing to the complexity of physiological and immunological derangements induced by cholelitiasis and obstructive jaundice , we also show that preoperative cholestasis and hepatic injury , might have a marked influence on perforin - related aspects of cytotoxic t and nk cells and final interpretation of data obtained by classic or laparoscopic cholecystectomy .
perforin-(p- ) related characteristics of cytotoxic t lymphocytes and natural killer cells were investigated in peripheral blood of patients subjected to open ( oc ; n = 23 ) or laparoscopic cholecystectomy ( lc ; n = 21 ) and healthy controls ( n = 20 ) . blood samples were obtained preoperatively and 24 hours after the surgeries , and the data were correlated with the intensity of cholestasis and concomitant inflammation , determined by functional hepatic tests . 
 postoperative differences were found to be minimal : oc decreased only the percentage of cd56 + cells , while lc decreased the fraction of cd8+p+ cells and augmented the mean fluorescence intensity of p in cd56 cells . 
 patients elected for oc had , however , higher preoperative numbers of total p+ , cd3+p+ , and cd4+p+ cells than patients elected for lc and healthy controls , while both groups of patients , preoperatively , had lower fraction of cd16+p+ and cd56+p+ cells . 
 these changes were in high correlation with blood concentrations of crp , ap , and alt , emphasizing the link between the preoperative cholestasis and inflammation and p - dependent cytotoxic mechanisms .