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J Clin Pathol 1982;35:972-979

Monocyte function in cirrhosisGREG HOLDSTOCK, B LESLIE, S HILL, AR TANNER, RALPH WRIGHT

From the Professorial Medical Unit, Level D, S Laboratory and Pathology Block, Southampton GeneralHospital, Southampton S09 4XY

SUMMARY Monocyte function has been studied in a total of 51 patients with biopsy-provencirrhosis and 35 controls. There was significantly reduced monocyte spreading (p < 0.05),chemotaxis (p < 0.02), bacterial phagocytosis (p < 0.05) and bacterial killing (p < 0.02) in thecirrhotics compared to the controls. Monocytes from patients with cirrhosis producedsignificantly less of the lysosomal enzymes N-acetyl f-glucosaminidase and ,8-glucuronidase thanthose obtained from the controls (p < 0.02). There was no significant difference in the number ofmonocytes obtained, the number of macrophage precursors, and the nitro-blue tetrazoline(NBT) reduction between the cirrhotic and the controls. The reduced function appeared to bemainly due to a circulating inhibitory factor and could be corrected by incubation of the cirrhoticcells in serum from control subjects. The response of monocytes from patients with cirrhosis didnot differ from the controls in their response to added endotoxin or latex particles suggesting thatthey are capable of a normal response in the absence of the inhibitory factor. Paired specimens ofportal and systemic serum were collected from patients with no evidence of liver disease undergo-ing vascular surgery. When added to normal human monocytes the portal serum caused asignificant reduction in bacterial killing (p < 0.02) and chemotaxis (p < 0.05) compared to resultsobtained in the paired systemic serum. Mixing experiments suggests the presence of an activeinhibitor in the portal serum.The results suggest that monocyte function is reduced in cirrhosis apparently due to a serum

inhibitor which may have originated from the portal vein. The abnormalities may account in partfor the increased susceptibility of these patients to infection.

A variety of defects in the cellular immune systemhave been described in liver disease, but the litera-ture concerning monocyte function in cirrhosis isscarce. The predisposition of patients with alcohol-ism and cirrhosis to infection'-3 is well recognised,and alcoholics may show varying degrees ofleucopenia4 and leucocyte functional derangement.5Von Epps and coworkers6 showed that inhibitors toboth leucocyte and monocyte chemotaxis was pres-ent in 50% of patients with alcoholic liver diseaseand that the severity of the defect correlated withserum IgG and particularly IgA concentrations.DeMeo' also showed chemotactic inhibitory sub-stance activity in serum from 19 of 22 patients withalcoholic liver disease, at least three differentchemotactic inhibitors being present. Hassner8demonstrated reduced phagocytosis and killing ofCandida albicans in patients with cirrhosis andsuggested the presence of a serum inhibitory factor.On the contrary Ganguly et a19 suggested that the

Accepted for publication 26 January 1982

production of acid hydrolase enzymes from mono-cytes was increased in cirrhotics, suggesting that thecells were "activated". These studies of monocytefunction in cirrhosis are of interest not only becausethey may account for the increased susceptibility ofpatients to infection but also because they may be anindicator of Kupffer cell function.'0

It has been postulated that, in cirrhosis, antigensabsorbed from the gastrointestinal tract may not beremoved by the failing liver either through shuntingof portal blood, or because of impairment of Kupf-fer cell function. These antigens may then resultin continuous immunostimulation leading toimmunosaturation and a reduced response tofurther superimposed infection." 12 These antigenscould be similar to the inhibitory substance dis-cussed above.The purpose of this paper is to try to define more

accurately monocyte defects in patients with cir-rhosis and to investigate the possibility of an over-spill phenomenon contributing to the abnormalitiesfound in cirrhosis.

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Monocyte function in cirrhosis

Patients and methods

PATIENTSOnly patients with biopsy-proven liver disease werestudied. Where indicated below, and where numberspermitted, the patients were classified as "active" or"quiescent" on the basis of the biopsy, biochemical,and clinical assessment. The former were mainlyin-patients, the latter out-patients. Age and sex-matched controls were selected from hospital staffand from patients attending a day-ward for minorsurgery. A standard full blood count and differentialwhite count was performed. Eleven alcoholicpatients with no evidence of hepatitis or cirrhosis onbiopsy and six patients with obstructive jaundicewere also studied. All studies were approved by thedistrict ethical committee.

CULTURE CONDITIONSA mixed mononuclear cell preparation (MMC) con-taining approximately 30% of monocytes wasobtained from fresh venous heparinised (10 U/ml)blood by density centrifugation following layeringon to lymphocyte preparation medium (FlowLaboratories, UK). This was then washed threetimes in Hanks' buffered salt solution (HBSS). Amonocyte-enriched preparation (MEC) wasobtained by adherence to plastic Petri dishes for twohours at which time nonadherent cells wereremoved. The adherent cells were then washed andharvested using a rubber policeman and thenwashed again. Viability was confirmed by exclusionof trypan blue. Cytospin preparations were stainedwith either Giemsa-Wright or non-specific esteraseand the MEC preparation was confirmed to containapproximately 90% monocytes. Any preparationwith a viability of less than 90% or which containedless than 80% of monocytes was discarded.

TESTS OF MONOCYTE FUNCTION

Macrophage precursorsThe macrophage precursor assay described by Cur-rie and Hedley'3 was used. In brief, MMC (2 x106/ml) in RPMI 1640 (+25 mmol HEPES andantibiotics) containing 50% autologous serum wasadded in 100 ,ul volumes to the wells of a plasticmicroplate. Plates were incubated at 370 in 5% CO2in humid air for seven days, when the wells werewashed free of unattached cells with HBSS and 50,ul of a 0.01 M citric acid solution containing 1:2000crystal violet added to each well.'4 The plates wereallowed to stand for 30 min at room temperature atwhich time the detached nuclei were counted in ahaemocytometer. This assay is thought to indicatethe number of circulating monocytes present which

have the capacity to mature into tissue mac-rophages.

Monocyte spreadingSpreading was measured using the methoddescribed by Territo and Cline.'5 In brief, 0-5 ml of a4 x 101 cell/ml MMC preparation was placed overglass coverslips in sterile plastic multiplates (LuxScientific Corporation). After 1, 24, and 48 hcoverslips were removed, gently washed in HBSSand fixed in 2% buffered glutaraldehyde for 5 min.The coverslips were then inverted on to oil drops inglass slides, taking care to avoid drying. The longestaxis of the cells was measured using phase-contrastmicroscopy with an oil-immersion objective lens(each reading mean of 20). This ability to spread isincreased when the monocytes are "activated" andis probably a measure of cell maturity.

NITRO-BLUE TETRAZOLIUM REDUCTIONThe redox dye nitro-blue tetrazolium (NBT) isreduced by the action of NADPH oxidase. The pro-duct is the insoluble coloured crystalline formazan.The assay is therefore an indirect measurement ofthe hexose monophosphate shunt. The methoddescribed by Hedley and Curry,'6 itself an adapta-tion'7 was employed. In brief, 150 ,ul of a MMCsuspension containing 106 mononuclear cells wasplaced in 2 ml polypropylene tubes. As a phagocyticstimulus, 50 ,ul of 0 79 ,tm latex polystyrene beads(Sigma) diluted in 1/100 in RPMI 1640 was addedand 50 ,ul of RPMI 1640 added to control tubes.After incubation at 37°C for 15 min, 25 ml of a 4nmol solution of NBT (Sigma) in 314 nm sucrosewere added. After a further one hour incubation,the reaction was stopped by adding one drop of 0 1N HCI to the tubes. The formazan was then ex-tracted by adding 500 ,l of Dioxan to the cell buttonand incubation at 70°C for 20 min. After cen-trifugation at 2000 g for 15 min, the clear super-natant was read at 520 cm on a spectrofluorometer,using Dioxan as a blank. A standard curve wasprepared by reducing doubling dilutions of NBTwith 150 nmol of ascorbic acid.'8

PHAGOCYTOSIS AND BACTERIAL KILLING OFSTAPHYLOCOCCUS AUREUJSThe method used was based on that described byTerrito and Clinel5 with minor modifications.'9 20The method below produced the most reproducibleresults in preliminary studies. Staph aureus was cul-tured overnight in a brain/heart medium and dilutedto a final concentration of approximately 2 x 106bacterial colony-forming units (CFU) per ml. Thiswas divided into aliquots, snap-frozen and stored at- 70°C for later use. For phagocytosis, 0-125 ,ul of a

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MMC preparation in a concentration of 4 x 106 cellswas added to 0*125 ul of autologous serum and0. 125 Al of bacterial suspension in a 12 x 75 mmplastic tube. After incubation at 37°C for one hour,a cytospin preparation was prepared and thenumber of bacteria ingested by 100 monocytescounted (mean of five readings). To measure bacter-ial killing 200 ,u of bacterial solution was put in a 12x 75 mm plastic tube. To this was added 200 Alautologous serum and 500 ,lI of a mixed mono-nuclear cell preparation containing 4 x 106 cells/ml.This was incubated for one hour at 37°C, sonicatedto disrupt cells and release bacteria (confirmedmicroscopically) and diluted 1/1000. The number ofCFU/ml was then measured by culturing on blood-agar plates for 24 h. The percentage killing was cal-culated from the equation: % killing = A - B/A,where A is CFU/ml for the control cultures-that is,tube containing only bacteria and no cells, and B isCFU/ml count for tube containing bacteria and cells.

CHEMOTAXISChemotaxis assays were performed by a modi-fication -of the Boyden chamber technique2' asdescribed by Wilkinson.22 Serum chemotactic activ-ity was produced by incubating fresh frozen humanAB serum with zymosan (Sigma Chemical Co 0-5mg/ml serum). A mixed mononuclear cell prepara-tion of 1 x 106 cells in 0*5 ml was added to the topchamber with no added serum. Distance moved bymonocytes through an 8 ,um millipore filter wasmeasured after incubation at 37°C for 4 h in a CO2incubator using the leading front counting techniqueas described by Zigmond and Hirsch.23 The mean of10 readings was recorded. An assessment of randommovement was obtained by the measurement ofmigration of mononuclear cells exposed to RPMI1640 alone (containing no zymosan-treated serum).This measurement was subtracted from the move-ment induced by the zymosan-treated serum toobtain the measurement of chemotaxis.

ACID HYDROLASE PRODUCTIONProduction of the acid hydrolase N-acetyl-glucosaminidase (NAG) and f3-glucuronidase (,fG)was measured using the methods described in detailelsewhere.9 HEPES-buffered RPMI 1640 supple-mented with 10% fetal calf serum (SRPMI 1640)was used for cell maintenance to each of six wells ofa sterile plastic multiplate (Lux Scientific Corpora-tion) monocytes enriched cells (1.5 x 106) inSRPMI 1640 were added and allowed to adhere tothe surface by incubation at 37°C in a 5% carbondioxide fully humidified atmosphere for two hourswith occasional agitation. After incubation, super-natant was discarded and three of the wells were

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filled with 1*5 ml of an endotoxin solution and threewith S-RMPI 1640 alone. The plates were incubatedfor a further two hours and the supernatants dis-carded, the cells washed three times in HBSS toremove the endotoxin and refilled with 1-5 ml ofS-RPMI 1640. Following a further 24-hour in-cubation, supernatants were collected from eachwell, centrifuged to remove any contaminating cells,and stored in plastic tubes at - 20°C. Adherent cellswere removed from the plastic surface of each wellwith a rubber policeman into 1-5 ml of sterileice-cold distilled water and the cell suspension wasrapidly frozen and thawed eight times to lyse themonocytes. It was not possible to carry out a cellcount before lysis, and we have assumed that the cellnumber after incubation bore a constant relation tothe starting inoculum in each case. The total proteinconcentration of cell lysate was estimated by themethod of Lowry et a124 and was used as an index ofcell mass.25 A sensitive assay for NAG and f3G wasdeveloped from the spectrofluorometric method ofBeutler et a125 as described previously.9 The endo-toxin solution Salmonella typhosa 0901 (DifcoLaboratories) was prepared in 0*85% sodiumchloride at a concentration of 1 mg/ml and dilutedwith RPMI 1640 medium to give a working concen-tration of 50 ,ug/ml.

FURTHER EXPERIMENTSMonocytes from a normal human volunteer wereincubated at 37°C for four hours in paired specimensof serum (20%) from the controls and from cirrho-tics. The cells were then washed three times prior tomeasurement of chemotaxis and bacterial killing.In these experiments the bacterial killing andphagocytosis was performed in fetal calf serum.In further experiments chemotaxis and bacterial kil-ling were measured on cells from cirrhotic patientsand incubated with control serum (conditions asabove) and compared to the results obtained bythose incubated in autologous serum. To investigatethe overspill phenomenon, paired specimens of por-tal serum and venocaval serum were collected.These were obtained from patients undergoing vas-cular reconstructive surgery. None of these patientshad clinical or biochemical evidence of liver disease.The effect of the paired serum specimens was meas-ured on chemotaxis and bacterial killing propertiesof monocytes from healthy volunteer donors(methods as for studies comparing cirrhotic and con-trol serum).

STATISTICSStudent's t test was used throughout. The Mann-Whitney U test was also used for comparing non-parametric data. Probability values of less than 5%

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Table 1 Numbers ofmonocyte and glass adherent cells in patients and controls (mean + SEM)

No of subjects Absolute No of monocytes No ofglass adherent cells recoveredin blood film (nolml) from 50 ml blood x 106

Controls 35 538 + 42 7-0 + 3.4Chronic active hepatitis 10 594 - 54 6-0 + 4-5Alcoholic cirrhosis 25 512 + 48 6.6 + 4.5Alcoholic non-cirrhotic 11 594 + 84 8-2 - 5-01All other cirrhotics 16 524 - 34 5 37 + 6-7Obstructive jaundice 6 582 + 39 7.9 + 5-6All cirrhotics 51 528 ± 56 6-1 + 5-6

were considered to be significant. All assays wereperformed in triplicate and were coded and readwithout knowledge of the patients' clinical details.The results are expressed as mean + SEM. Interas-say variability was less than 15% for all the assays.

Results

Not all the tests of monocyte function were meas-ured concurrently. Table 1 shows the total numberof patients studied, the nature of the liver disease,and the total number of monocytes in the blood filmand number of adherent cells recovered for eachgroup. There was no significant difference betweencontrols and cirrhotics. Although the number ofmacrophage precursors was slightly reduced in thecirrhotics (9-2 ± 3.6 x 104/ml for the controls, and8-8 ± 4*2 x 104/ml for the cirrhotics, n = 16), thedifference did not reach statistical significance. Theresults for monocyte spreading in controls (n = 15)were 12*4 gm ± 2-4, 17*4 gm ± 2*2, and 19-4 ,um ±3*4 at time 0, 24, and 48 h respectively. Correspond-ing values for the cirrhotics (n = 15) were 12*8 gm± 2.7, 14-4 ,um ± 1.8, and 16-8 ,um + 3*4. The differ-ences were small and only reached statisticalsignificance at 24 h (p < 0.05). The reduction ofNBT was 12-2 ± 4-0 x 10- 5 per unstimulatedmonocyte, and 14-2 ± 3-6 x 10-15 per monocytestimulated with latex for the controls, and 9*6 ± 3-8X 10-'5 and 12-4 ± 4.2 x 10-15 per monocyte forthe cirrhotics. Thus, NBT reduction tended to belower in the cirrhotics, but again the difference didnot reach statistical significance. Both controls andcirrhotics responded to the latex beads in a similarfashion and the increase expressed as a percentagewas not significantly different in the two groups.

Results of phagocytosis, bacterial killing andchemotaxis are shown in Table 2. Both phagocytosisand chemotaxis were significantly reduced in the cir-rhotics. Although reduced, the overall difference inkilling between the cirrhotics and the controls didnot reach statistical significance, but, comparingthose with active disease (n = 10) there was asignificant reduction (29% + 3-8 v 40% ± 3.6, p <0.02), suggesting that the more severe the disease,

Table 2 Results ofchemotaxis, bacterial phagocytosis andkilling in cirrhotics and control patients (mean + SEM)

Chemotaxis (/pm) Phagocytosis Killing (%)(n = 24) Nol100 cells (n = 30)

(n = 16)

Cirrhotic 12 + 6 123 ± 18-4 34 - 4patients p < 0-02 p < 0-05 NSControls 39-2 + 3-8 149 + 28-4 40 - 3-6

the greater the loss of function. In the case of bacter-ial killing, in a separate group of 15 patients, incuba-tion of cells from cirrhotic patients in control serumresulted in marked improvement of function com-pared to the results in autologous serum 41% + 4-2and 12% + 6 respectively (p < 0.02) suggesting thatthe difference is due to serum inhibitory factors.Similarly for chemotaxis there was a significantreduction in the cells from cirrhotics when preincu-bated in autologous serum, but if these were prein-cubated with control serum chemotaxis was notsignificantly different from the controls (34 Am ±5-6 v 37-4 ,um ± 4.2).The result for the production of NAG and f3G

are shown in Tables 3 and 4 which show values insupernatants and the cell button respectively anddemonstrate the results of stimulation withendotoxin. In general, activities in supernatant andcells were decreased in cirrhotics compared to thecontrols. Differences were more marked for theNAG than for ,8G, where very low activities wererecorded throughout. Results from 11 alcoholicpatients without significant liver disease are alsoshown, and these values were not significantly dif-ferent from control values. Table 5 shows the resultsfor enzyme production divided into patients groupedon clinical grounds. Those with the severest disease,especially those with alcoholic hepatitis, were foundto have the lowest activities, whilst those with quies-cent disease were found to have near normal values.The small group of patients with cirrhosis onsteroids were found to have slightly increased levels,although not statistically significant, and this groupincluded most, but not all, the patients with chronicactive hepatitis. Studying the chronic active group as

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Table 3 Enzyme release by human monocytes measured in supernatant (mean (nmoll,g prot/h) +SEM)

No of NAG /3Gsubjects

Unstimulated Stimulated Unstimulated Stimulated

Controls 35 2-3 (026) 2-5 0-291) 0-13 (0.06) 014 (0.012)All cirrhotics 35 155 (03)* 1.8 0.4)* 0.099 (0.03)** 0-124 (0.09)

Chronic active 10 2-4 (0.4) 2-4 0-4) 0-11 (.04) 0-12 (0.02)Alcoholic + cirrhosis 15 1-2 (0.119)*** 1-6 0-28)** 0.079 (0.005)* 0-091 0-009)

- cirrhosis 11 2-0 (035) 21 0.39) 0-076 (0-01) 0-09 0.016)Other cirrhotics 10 1-2 (0.15 * 16 0.25) 0-12 (0-03) 0-18 0.05)Obstructive jaundice 5 19 (0-24 2-2 0-28) 0-16 (004) 017 0-02)

*p < 0-05 **p <0-02 ***p < 0-01.

Table 4 Enzyme cell content of monocytes (mean (nmolIl,g protlh) ± SEM)

No of NAG /Gsubjects

Unstimulated Stimulated Unstimulated Stimulated

Controls 35 0-48 (0-06) 0-52 (0-07) 009 (0.1) 011 (0.02)All cirrhotics 35 036 (0.06)** 0-46 0-06) 006 (0-01)* 0085 (0.02)Chronic active 10 0 49 (0-28) 064 0-09) 0-06 (0.01) 0-06 (0.05Alcoholic + cirrhosis 15 0-23 0-05)** 0-26 0-05)** 0-06 (0-01)** 0-07 (0-01

- cirrhosis 11 0-43 0-08) 0-45 (0.11) 007 (0-01) 008 (0.01Other cirrhotics 10 0-39 0-08) 0-69 (009) 0-06 (0.01)* 0-06 (0.01Obstructive jaundice 5 0 25 0-08 0-66 (0-28 0-09 (0-015) 0-12 (0-05

*p < 0.05 **p <0-02 ***p < 0-01.

Table 5 Production ofenzymes by human monocytes (unstimulated) in patients grouped by severity ofthe disease (mean(nmoll,ug protlh) + SEM)

No of NAG 8Gsubjects

Supernatant Cells Supernatant Cells

Controls 35 2-3 (0-35) 0-48 (0-07) 0-13 (0-02) 0-09 (0-01)Acute alcoholic hepatitis 4 1-3 (0.36)* 0.55 0.-1) 0-08 0-017) 0-094 (0.03)Active cirrhosis 10 1-34 (0.132)** 0-29 0-06)** 0.079 0-06)* 0-057 (0.024)Quiescent cirrhosis 17 1-95 (0.27) 0-446 0-115) 0-132 0-029) 0-069 (0-012)Cirrhosis on steroids 8 2-67 (0-5) 0-65 (0-25) 0-113 (0-016) 0-086 (0.02)

*p < 0.05 **p < 0-02.

Table 6 Effect ofcontrol and cirrhotic serum; portal and systemic specimens; and mixing experiments on monocytesobtained from a normal volunteer (mean + SEM)

Chemotaxis (pim) Bacterial killing (%o)Control serum (n = 20) 28-4 + 3-2 17-4 ± 3-8Cirrhotic serum-(n = 20) 14 ± 4-5 (p < 0.05) 7-5 3-5 (p < 0-02)Mixture (n = 15) 16 ± 54 (p < 0-05) 8-4± 4-2 (p < 0-05)

Systemic serum (n = 18) 49-2 ± 2-7 65 + 8-4Portal serum (n = 18) 45-8 ± 22 (p < 0-05) 29-2 ± 42 (p < 0-02)Mixture (n = 15) 46-2 ± 2-4 (NS) 34-6 ± 6-8 (p < 0-05)

The systemic/portal serum results were performed at different times and on different cells than the control/cirrhotic serum. Mixture refersto pooling of the relevant samples using equal volumes of each.

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a whole, levels were not significantly below normalvalues.A comparison of the effects of paired specimens

of cirrhotic and control serum is shown in Table 6.Both chemotaxis and killing are significantlyreduced by preincubation in cirrhotic serum com-pared to the controls. Also in this Table are shownthe values for preincubation of normal monocyteswith paired specimens of portal and systemic serum.Portal serum also results in significantly reducedchemotaxis and killing compared to the systemicserum. Mixing of cirrhotic with control and portalwith systemic serum in equal volumes resulted inreduced chemotaxis and killing in the same range asthose obtained for the cirrhotic and portal speci-mens respectively.

Discussion

We have attempted to test various aspects of mono-cyte function in patients with cirrhosis and havedemonstrated that this function is generally reduced.Although the changes are modest and do not alwaysreach significance, results in cirrhotics are consis-tently reduced. Changes reached significance forchemotaxis, bacterial killing, monocyte spreadingand bacterial phagocytosis. The number of mono-cytes present in cirrhotics was not different from thecontrol population, but the results could be inter-preted to suggest that the ability of these cells tomature into macrophages is less in cirrhotics than inthe controls. Failure to show more clearcut differ-ences between the cirrhotics and controls may bedue to the inadequacies of currently availabletechniques. This reduced function appears to bemainly due to a serum inhibitor as incubatingcirrhotic cells with normal serum resulted in someimprovement in their function. Experiments inmixing cirrhotic and control serum showed thatmonocyte function was still reduced, and this wouldsuggest the presence of an active inhibitor ratherthan a deficiency situation. Unfortunately thenutritional status of the patients was not assessed. Ithas previously been shown that there is defectivebacterial opsonisation in patients with cirrhosis,2627but previous work has differed in respect to thenature of this defect. Some have found, as we have,that there appears to be an active inhibitor ofchemotaxis and phagocytosis present.6 8 Others,based on the results of mixing and dilution experi-ments, have suggested that a relative deficiency stateexists, possible of complement, and that thisaccounts for the reduced opsonisation in cirrhotics.26In our view the reduced phagocytic function is un-likely to be simply due to a defect of opsonisation, asafter preincubation with the appropriate serum the

actual phagocytosis was performed in fetal calfserum in these experiments. Similarly if this was adefect of opsonisation the defect would not bepresent on mixing the two sera.Our results would suggest that the monocyte

defect is secondary to the cirrhosis and not depen-dent on the underlying aetiology as it was present inall groups of cirrhotics to some extent. The enzymeproduction by the group of alcoholic patients, inwhom there was no cirrhosis or hepatitis, was alsonot different from the controls. In chronic activehepatitis patients with cirrhosis, monocyte functionwas also reduced but all but one patient was takingcorticosteroids, which, in the short-term at least, areknown to depress monocyte function.28 Long-termcorticosteroid treatment has less effect on monocytefunction, but we cannot exclude the possibility thatthe results obtained in our patients with chronicactive hepatitis may be artificially lowered by thecorticosteroid treatment, and that this may explainsome of the differences obtained between ourresults and those previously obtained in a smallgroup of patients in our laboratory.9

While most of the evidence suggests that thereduced function is due to the presence of an in-hibitory factor, the NAG and 8G productionresults might suggest that the cells themselves areabnormal, as these were performed in fetal calfserum. The production of these enzymes probablymirrors the ability of the monocytes to differentiateinto macrophages29 and further evidence to showthere is a relative failure of maturation of these cellsis obtained from the spreading experiments. Hass-ner recently reported8 that treatment of cirrhoticmonocytes with trypsin (to remove all traces ofserum factors) resulted in a return of monocytefunction to normal. Thus, although the cells werewashed carefully, it is probable that these defects inenzyme production could also be accounted for byan effect of a serum factor carried over by the cells.However, we cannot exclude the possibility thatthere is a shift in the population of circulating mono-cytes in cirrhosis, although the findings of normalnumbers of circulating monocytes which functionnormally in control serum makes this unlikely.

For enzyme production and NBT reduction, themonocytes were studied before and after stimulationeither by endotoxin or latex and, in both cases, theresponse of the cells to this stimulation was notsignificantly different from that of the controls.Thus, the theory of hyperstimulated cells becomingimmunosaturated and unable to react to freshstimuli has not been substantiated and it wouldappear that these cells are capable of a normalresponse, at least in vitro. Endotoxaemia may becommon in cirrhotics3031 and assuming that the in

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vivo effects are similar to the in vitro effects, a stateof "monocyte activation" might have beenexpected. The relevance of the concept of the acti-vated macrophage32 is unknown but it is a term usedto indicate a state of increased activity of the cellswhich occurs on exposure to various stimuli includ-ing endotoxin. Contrary to our expectation itappears that in cirrhosis the cells are less "activated"and that the serum contains "deactivating" sub-stances. Activated macrophages also excrete col-lagenase33 and thus this mechanism may be impor-tant in the instigation or perseverance of increasedcollagen deposition in the liver and hence thedevelopment of cirrhosis.We have shown that normal cells exposed to por-

tal vein serum show reduced chemotaxis and bacter-ial killing compared to those exposed to simultane-ously taken systemic serum specimens. The mixingexperiments would also suggest that there is anactive inhibitor present. Thus, rather than themonocyte defect being explained on the basis of aconstant stimulation by antigens resulting in a fail-ure to respond to fresh stimulation, our results wouldsuggest that inhibitory substances (probably gut-related antigens) bypass, or are not sufficientlyremoved by, the failing Kupffer cells in the cirrhoticliver. We have further investigated this by measur-ing immune complexes and endotoxin in the portalsytemic serum and comparing them to the effect ofthe individual specimen on the monocyte responses.These studies suggest that it is immune complexesrather than endotoxin which have the most markedeffect (Holdstock et al submitted for publication).

Ferluga34 has suggested that monocytes, whichare presumed to be Kupffer cell precursors, may beimportant in certain types of liver damage. He hasdescribed an animal model where mice, primed withCorynebacterium parvum and exposed toendotoxin, developed acute liver failure. He sug-gests that monocytes recruited into the liver by theCorynebacterium parvum may be activated by theendotoxin to release potentially toxic substances. Hefurther suggests that the same mechanism may beoperative in hepatitis. We have recently providedfurther evidence for this from in vitro studies inrats35 but contrary to our original expectation9 thatenzyme release from monocytes might be increasedin cirrhosis, and thus a possible indicator of activeliver damage, the finding that enzyme release isreduced does not support this theory of hepatocytedamage in the patient groups studied. Crucial to thisis the unanswered question regarding the relation ofperipheral monocyte function to that of Kupffercells. If such a relation exists our findings suggestthat Kupffer cell function is reduced in cirrhotics.Thus the Kupffer cells may be unable to protect the

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hepatocyte from further damage. This finding is inkeeping with recent in vivo studies of Kupffer cellsactivity36 suggesting reduced function in cirrhosis.

In conclusion, we have demonstrated wide-ranging defects in monocyte function in patientswith cirrhosis. This appears to be secondary to thepresence of cirrhosis and is probably not of primaryimmunopathogenic importance. The abnormalitymay be due to the presence of an active inhibitorwhich possibly originates from the portal venous sys-tem due to failure of the filtering mechanism of thenormal liver. The defects in monocyte functiontogether with previously shown defects in neutrophilfunction and other abnormalities including defectiveserum bactericidal activity," may all play a role inthe susceptibility of these patients to infection. It ispossible that nutritional deficiencies38 in part explainthe abnormalities, but this would not seem toexplain the presence of an active inhibitor sub-stance.

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Requests for reprints to: Dr GE Holdstock, ProfessorialMedical Unit, Level D, S Laboratory and PathologyBlock, Southampton General Hospital, Southampton S094XY, England.

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