European Journal of Clinical Investigation (1977) 7,571-577

Blood polymorphonuclear dysfunction in patients with alcoholic cirrhosis EVARIST FELIU, MARIE-ANNE GOUGEROT, JACQUES HAKIM,* ELISABETH CRAMER, CHRISTIAN AUCLAIR, BERNARD RUEFF & PIERRE BOIVIN, Laboratoire Central d’Immunologie et d’Hematologie, Hopital Bichat, and Service d’Hepato-Gastroenterologie, Hopital Beaujon, ERA 573 (C.N.R.S.), U 160 (I.N.S.E.R.M.), Universite Paris VII, France Received 24 November 1976 and in revised form 10 June 1977

Abstract. Polymorphonuclear leucocyte function was investigated in twenty patients with alcoholic cirrhosis and three patients with cryptogenic cirrhosis. Bacterial ingestion, oxygen-dependent bactericidal capacity, and chemotactic response were measured. Serum dependent abnormalities were common; they included deficiencies of ingestion and of all subsequent oxygen-dependent metabolic events (three patients), all oxygen-dependent metabolic events (one patient), cytochrome c reduction and iodination deficiencies (six patients), isolated cytochrome c reduction deficiency (ten patients), and chemotactic deficiencies (fourteen out of eighteen patients). Serum-independent abnormalities were much less common; they included increased ingestion rate (four patients), decreased stimulated reduction of nitroblue tetrazolium (three patients), and decreased myeloperoxidase content (eight patients). Polymorphonuclear leucocyte abnormalities are frequent in cirrhosis and may account in part for increased susceptibility to infection in that disease. Key words. Leucocytes, metabolism, cirrhosis.

Introduction

Patients with liver cirrhosis show an increased susceptibility t o infection [l-51. Human defence against pyogenic infections depends on the humoral immune system and the phagocytes (polymorphonuclear leucocytes, monocytes and macrophages). Previous studies have reported abnormalities in the humoral immune system [6- 121 ; however, these abnormalities are rarely sufficient to compromise defence mechanisms [7,13]. Polymorphonuclear blood leucocytes (PMN) are important in the host’s defence [14, 151 and three of their functions are important in this: mobilization to the infection site [16], ingestion of the infectious agents [I71 and intraleucocytic microbicidal activity [ 181. The most potent PMN bactericidal system is oxygen-dependent Correspondence: Dr Jacques Hakim, Laboratoire Central d’ Immunologie et d’Hlmatologie, HBpital Bichat, 170 Boulevard Ney, 75877 Paris Cedex 18, France.

[19]. Recently, defective leucocyte chemotaxis due t o serum factor(s) has been shown in cirrhotic patients [7, 20, 211. In vivo leucocyte mobilization has also been found defective in severe alcoholic liver cirrhosis [22]. It has been suggested that this phenomenon may in part account for the increased susceptibility of cirrhotics t o infection. We have looked for abnormalities in the PMN of patients with cirrhosis. Special attention was given to the ingestion phase and oxygen-dependent metabolic events: O2 consumption, superoxide anion (0;) and hydrogen peroxide (H202)production, and iodination. Patients and Methods

Patients Twenty-three patients (sixteen male and seven female; aged 36-69 years) with alcoholic liver cirrhosis (patients 1-20), or cryptogenetic cirrhosis (patients 2 1- 23) associated with chronic active hepatitis in the case of one patient (patient 21) were studied. Diagnosis was established by liver biopsy andlor laparoscopy. All patients had a normal number of circulating PMN. None had bacterial infection at investigation and none had received any drug known t o modify PMN activity. All studies were done after cessation of alcoholic ingestion for at least 10 days. Forty healthy male and female blood donors or persons from the hospital staff, aged 30-50 years, served as controls. The purpose of this study was explained to the patients and the volunteers. They readily agreed to take part. Methods Preparation of serum. Venous blood samples were allowed to clot at room temperature for 2 h and serum separated by centrifugation. A fraction of each serum was used and the remainder was stored in 1 ml aliquots at - 80°C until further testing. Ten control AB sera were pooled and stored in 1 ml aliquots at - 80°C. Storage did not modify the activities measured.

571

572

EVARIST FELIU et al.

Serum immunoglobulins and complement. Serum immunoglobulins (IgG, IgA and IgM) were measured by radial immunodiffusion. Commercially prepared plates (Behring) were used. Total serum haemolytic complement was determined according to Kabat & Meyer [23]. Complement components (C3, C4 and C3 proactivator) were measured by radial immunodiffusion (Behring. plates). Preparation of leucocyte suspension. Leucocytes from patients and controls were isolated as described previously [24,25] from heparinized (10 U/ml of blood) venous blood. The blood was mixed with Dextran T 500 (Pharmacia Uppsala, Sweden) and allowed to settle. After sedimentation of most of the red blood cells, the remaining red blood cells in the leucocyte-rich supernatant were lysed with ammonium chloride. Following three centrifugations and washings with Krebs-Ringer phosphate calcium free solution (KRP) a differential count was made. The final cell suspension was adjusted to l o 7 PMN/ml. It contained more than 80% PMN and contaminating cells were mainly lymphocytes. Cell viability was assessed by trypan blue exclusion [26]. This leucocyte suspension was used for all PMN function tests, chemotaxis excepted, The PMN used for chemotaxis were isolated according to Boyum [27, 281 : the PMN and red blood cells, first separated from the other blood cells by the Ficoll-triosil technique [27] were layered above a Dextran-radioselectan solution [28] in order to remove most of the red blood cells. The PMNrich supernatant was then treated as described above. PMN function tests performed in the presence of serum. The following tests were performed in either autologous serum or pooled AB serum (control serum): (1) Ingestion rate was measured by a modification of Mandell’s [29] technique: heat-killed non-virulent 14Clabelled Klebsiella after appropriate opsonization in the patient’s serum or control serum were used; the Klebsiella selected did not sedimentate at 350 g for 8 min. The incubation medium contained 0.5 ml of the PMN suspension (5 X 10S/ml) and 0.5 ml of the selected Klebsiella suspension in KRP (5 X lo7microorganisms/ml) containing 20% serum. A concentration of 2 X lo8 bacteria/ml was achieved by dilution with KRP to an optical density of 0.400. Final counts were made in a Petroff-Hauser chamber to give a more accurate and reproducible stock suspension of 2 X lo8 bacteria/ml. Before and after tumbling the tubes (20 RPM) for 5, 10 and 15 min, the ingestion process was stopped with ice-cold acid-citratedextrose. Separation of non-ingested Klebsiella from the PMN was performed by three centrifugations and washings at 150 g for 6 min. It was checked microscopically that no Klebsiella were present outside the PMN. The cell buttons were digested with Soluene-350 (Packard Inst. Co), and counted in an Intertechnique (ABAC SL.40) scintillation counter. A standard curve was run in parallel with each experiment so that the relative radioactivity could be directly converted to number of Klebsieila ingested. As for the linearity of the ingestion rate

observed in the controls and patients, results expressed in terms of microorganisms associated per PMN in 10 min at 37”C, were the means of the results of the three incubation times. (2) Latex-stimulated histochemical nitroblue tetrazolium (NBT) reduction was performed according to Windhorst et al. [30] in the presence of 60%serum and of cyanide (1 mM). Results were expressed as the percentage of PMN which engulfed more than ten latex particles (histochemical latex test), and among these the percentage of those which reduced NBT (histochemical NBT test). (3) Cyanide-insensitiveO2 consumption of resting and zymosan-stimulated (Zymosan A from Saccharomyces cerevisiae) PMN and (4) H 2 0 2 production, were measured polarographically (Gilson oxygraph equipped with a Yellow Springs electrode) according to Kvarstein [31] and Takanaka & O’Brien [32], respectively. The incubation medium contained 10%serum, 1 mM cyanide and lo6 PMN/ml. Results were expressed in nanoatoms of O2 consumed and in nanomoles of H 2 0 2 produced per min and per lo6 PMN at 37°C. (5) Zymosan-stimulated Qi production was measured by cytochrome c reduction according to Curnette & Babior [33] as modified by Weening et al. [34]. The incubation medium contained 0.33 mg zymosan/ml, 80 pM cytochrome c, 10% serum and 2.0-2.5 X lo5 PMN/ml. Cyanide was absent in the incubation medium. No reduction of cytochrome c occurred when superoxide dismutase was added to the incubation medium. Incubation times were 0, 5, 10 and 15 min. The results expressed in nanomoles of reduced cytochrome c per rnin per lo6 PMN were the means of the results of the three incubation times. (6) The iodination test was performed at 37°C according to Pincus & Klebanoff [35], as modified by Hakim et al. [24]. The incubation medium contained 10% serum and either 20 or 100 p M iodide. Incubation times were 0, 1 0 and 20 min. The results, expressed in nanomoles of iodide converted to a trichloroacetic precipitable form per hour per lo7PMN, were the means of the results of the two incubation times. (7) Ozemotaxis was measured according to Cutler [36] and Nelson et al. [37]. Chemotaxis of control PMN in 20% serum taken from the patient or from the control, were measured in parallel. The attractant used was the supernatant of a Klebsiella culture. T h s supernatant was separated from the Klebsiella culture by centrifugation at 100 000 g for 30 min. A stock pool of attractant was prepared, sufficiently diluted to be ratelimiting in chemotaxis, and stored in small fractions at -880°C for further use. Just before use, one of these fractions was thawed and mixed with an equal volume of control or patients’ serum. Chemotaxis (attractant patients’s serum) results were expressed in percentages of the controls’ chemotaxis (attractant + control serum), run in parallel.

+

PMN function tests performed in the absence of serum. (1) Zymosan-stimulated quantitative NBT reduc-

POLYMORPHONUCLEAR DYSFUNCTION IN CIRRHOSIS tion was performed in the presence of 1 mM cyanide, according to Baehner & Nathan 1381. Incubation time was 15 min at 37°C. Results were expressed in nanomoles of NBT reduced per rnin per lo6PMN. (2) Myeloperoxidase (MPO) was histochemically evaluated in the PMN with benzidine. Each of the 200 PMN observed was scored as follows: 0, no activity; 1, activity present in a portion of the cytoplasm; 2, activity present in the whole cytoplasm; and 3, activity present in the whole cytoplasm and covering the nucleus. MPOscores were expressed per 100 PMN. All measurements were performed in duplicate with appropriate blanks and standards.

573

Results

Function tests of PMN from patients with alcoholic cirrhosis: measurements performed in autologous serum (Table 1) The PMN of five patients (patients 1-5) showed no deficiencies. However, PMN from patients 3 , 4 and 5 had a high Klebsiella ingestion rate, and those of patient 5 a high O2 consumption, H202 production and iodination with a normal cytochrome c reduction (0;-measured production). The PMN of seven patients (patients 6- 12) showed an isolated deficiency of cytochrome c reduction (3.3 + 0.8 nanomoles per min per 106 PMN). PMN samples from patient 12 also revealed a high Klebsiella ingestion rate. The PMN of five other patients (patients 13- 17) showed a deficiency in both cytochrome c reduction (2.3 f 1.1 nanomoles per min per lo6 PMN) and iodination (7.62 f 1.68 nanomoles per hour per lo' PMN) while other oxygen-dependent function tests performed in the presence of cyanide were normal. Furthermore, the normal increase in iodination seen when 20 p~ iodide was increased to 100 p M in the incubation medium [24] was not observed. The PMN of patient 18

Statistical calculations. For each test a mean and a standard deviation has been calculated from a number of control-observations taken on different days. The comparison between the individual results and the mean of the controls were based upon the dispersion of this mean (t-test for unpaired data). Comparison of grouped results have been based on the t-test for paired data, since the experiments were run in parallel.

Table 1. PMN function in autologous serum in patients with alcoholic cirrhosis

Iodination (lodide)

Histochemical test Patients

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Controls Mean + 1 SD (No.)

ingestion rate (1)

Latex (2)

NBT (3)

0,

(4)

HZO, (5)

6.2 5.8 23.7** 17.1** 19.6** 6.8 8.6 5.0 5.1 10.0 8.8 17.7* 5 .5 7.6 4.3 9.2 4.4 11.4 2.5* 2.7*

97 97 99 100 91 97 97 98 97 92 97 97 78 98 94 98 98 86 81 95

77 84 76 97 72 81 97 77 85 83 79 81 69 85 87 71 72 51* 36* 61 *

13.5 15.0 16.4 16.3 24.9* 12.7 16.4 20.3 12.1 14.5 18.9 12.4 23.5 10.7 18.1 11.5 13.8 8.2* 7.1* 8.1 *

6.0 7.0 6.8 6.2 11.4* 5.6 7.9 6.8 4.8 6.2 6.8 4.8 8.5 4.9 6.3 4.5 5.3 3.2* 2.8* 3.1*

7.0

87 +7 (30)

76 +8 (30)

+ 2.5

* 1.1

(40)

(30)

+ 2.0 (25)

16.0

6.2

20 PM (71

100 pM (8)

1.8*

22.1 12.4 12.0 13.1 31.0* 14.4 13.5 13.3 11.6 22.8 22.3 16.9 6.2* 6.0* 7.2*

71 32 62 119 91 62 50 43 86 40 60 15" 9* O*

3.2* 3.6* 2.1 * 3.4* 2.6 *

8.8*

-

9.9* 8.6* 8.8* 11.3

81 102 36 42

0;

(6) 6.5 6.5 6.6 5.7 6.3 3.6* 4.1* 4.0* 4.0* 2.4* 2.3* 2.8* 1.0* 1.8*

7.0 + 1.3 (30)

(1) Number of associated microorganisms/lO min/PMN. (2) Percentage of PMN which engulfed latex particles. (3) Percentage of PMN containing blue-black formazan deposits calculated from the number of latex-containing PMN. (4) Stimulated 0, consumption in nanoatoms of 0, taken up per min and per lo6 PMN. Resting 0, consumption was in the control range 1.6 f 0.6. ( 5 ) Stimulated H,O, production in nanomoles/min/106 PMN. (6) Cytochrome c reduction (0: measured production) in nanomoles/min/106 PMN. (7) nanomoles/hour and/lO' PMN. (8) Percentage increase of iodination when iodide in the incubation medium was 100 pM. * and ** statistically significant decrease* or increase** within a 5% significance limit.

574

EVARIST FELIU et al.

showed normal ingestion but defective behaviour in all subsequent oxygen-dependent metabolic events including histochemical NBT reduction. PMN from patients 19 and 20 showed a decreased ingestion rate with deficiencies of all subsequent oxygen-dependent metabolic events, including histochemical NBT reduction.

Table 2. PMN function test in control serum in patients with

alcoholic cirrhosis

Patients

Function tests of PMN from patients with alcoholic cirrhosis: measurements performed in control serum (Table 2) PMN with cytochrome c reduction deficiency in autologous serum (patients 10- 15, 19 and 20) were found to reduce cytochrome c in the control range when measured in control serum. The increase of cytochrome c reduction (2.7 f 0.9 nanomoles per min per lo6 PMN) was significant (P < 0.001 ; t-test for paired data). PMN iodination measured in control serum was also increased (5.9 ? 3.2 nanomoles per hour per lo7PMN) significantly (P < 0.00 1; t-test for paired data) and found to be within the control range (four patients out of five studied) with PMN deficient in iodination when measured in autologous serum. PMN ingestion rate in control serum was found to be within the control range with PMN (patients 19 and 20) showing a decreased ingestion rate when measured in autologous serum.

10 11 12 13 14 15 18 19 20 Controls Mean f 1 SD (No.)

Ingestion rate (1)

0;

(6) 5.2 5.7 4.5 4.1 6.1 5.8

-

-

6.7 11.0

5.2 4.4

7 .O 2.0 (25)

* 1.3

7.0

i

(30)

Iodination (Iodide ) 20 gM (7)

16.8 11.8 8.9* 15.2 13.5

16.3 3.2 (401 f

Legend, see Table 1.

Table 3. Pmn function in the absence of serum in alcoholic cirr-

hosis

Function tests of PMN from patients with alcoholic cirrhosis: measurements performed in the absence of serum (Table 3) The quantitative NBT reduction was found to be decreased in the PMN of only two patients (patients 18 and 19). MPO-scores were found t o be decreased in the PMN of seven patients, and the deficiency involved all the PMN.

Function tests of control PMN: measurements performed in the serum from patients with alcoholic cirrhosis (Table 4) The serum from patients 4, 5 and 12 whose PMN showed a high ingestion rate in autologous serum (Table l), did not increase the ingestion rate of control PMN. On the contrary, the serum from patients 19 and 20, whose PMN showed a decreased rate in autologous serum (Table l), inhibited the ingestion rate of control PMN. The serum from patients 6 - 17 (whose PMN showed a decreased cytochrome c reduction in autologous serum) and from patients 4 and 5 decreased cytochrome c reduction of control PMN (P< 0.001 ;t-test for paired data). The serum from patients whose PMN showed a decreased iodination in autologous serum, decreased the iodination of control PMN (P < 0.001 ; t-test for paired data). The serum from patients whose PMN showed normal iodination in autologous serum, did not modify the iodination of control PMN. Chemotaxis of control PMN was significantly de-

Patients 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Controls Mean f 1 SD (No.)

Quantitative NBT reduction (1) 1.41 1.23 2.06 1.65 1.02 1.16 1.27 1.10 1.25 1.35 1.08 1.78 1.15 1.09 1.07 1.93 1.29 0.87* 0.1 3* 1.27 1.61 0.29 (33)

f

MPO score (2) 191 192 200 125* 170 218 91* 168 75 * 224 202 210 200 104* 198 207 234 101* 120* 97* 150 to 250t

For techniques used and expression of results, see Methods. (1) Zymosan-stimulated NET reduction, results expressed in nanomoles/min/106 PMN. (2) Results expressed per 100 PMN with a score ranging from 0 to 3 per PMN. * Statistically significant decrease within a 5% significance limit. t Control range.

POLYMORPHONUCLEAR DYSFUNCTION IN CIRRHOSIS Table 4. Function of control PMN in serum of alcoholic cirrhotic

creased by the serum of twelve of sixteen patients with alcoholic cirrhosis (Table 4).

patients Ingestion rate -

Patients 1 2 3 4 129 5 92 6 7 8 9 10 11 12 91 13 14 15 16 17 18 19 40* 20 so* Controls 100 Mean t 1SD t 12 (No.) (20)

Cytochrome c reduction (0:)

Iodination

Chemotaxis

92 107 105 79* 57* 64 * 68* 57* 74 * 47* 32*

96 102 99 107 87 105 99 106 96 102 89 92

53* 73* 73* 92 122 53* 55 * 53* 56* 53*

-

-

73* 74* 72* 79 * 76* 82* 108

71 * 100 80

??* -

59* 61* 48* 62* 75* 57* 64* 100 *8 (23)

PMN function tests in patients with cryptogenetic cirrhosis The patients’ PMN activities measured in autologous serum, control serum and in the absence of serum are reported in Table 5, together with the effect of the serum of these patients on controls’ PMN. The abnormalities observed were quite similar to those of patients with alcoholic cirrhosis. Discussion

Several previous studies [7, 20, 2 1] have demonstrated defective chemotaxis of blood PMN in patients with liver disease. Our study showed other dysfunctions of engulfment and of intraleucocytic oxygen-dependent bactericidal events in PMN from patients with alcoholic cirrhosis. Furthermore, our data suggest that PMN from patients with cryptogenetic cirrhosis displayed PMN abnormalities similar to those of PMN from patients with alcoholic cirrhosis. The PMN abnormalities observed were quite heterogeneous. They were mainly deficiencies dependent on serum of these patients. An argument in favour of the serum dependence of a deficiency is that the deficiency occurs when the PMN are studied in autologous serum and that it is corrected when the patient’s

-

58* 71 * 75*

100 11 (26)

100 t 7 (22)

575

t

Results are expressed in percentage of the mean of the activity measured in parallel with each assay, in three control serum. * Statistically significant decrease within a 5% significance limit.

Table 5. Results of function tests of the PMN from patients with cryptogenetic cirrhosis and effect of their serum on control PMN

Patients

21

I. Patient’s PMN activity measured in autologous serum: Ingestion rate (1) 1.1* 96 Histochemical test Latex (2)

1

NBT (3) 70 Stimulated 0, consumption (4) 7.9* H,O, production (5) 2.3* 0 ; : cytochrome c reduction (6) 2.4* 20 P M (7) 3.7* Iodination (Iodide) 83 100 p M (8) 11. Patients PMN activity measured in control serum: 8.8 Ingestion rate (1) 0 ; : cytochrome c reduction (6) 6.2 Iodination (Iodide) 20 p M (7) 5.8* 111. Patient’s PMN activity measured in the absence of serum (9) Quantitative NBT reduction 1.00 MPO score 14** IV. Control PMN activity measured in patient’s serum (10): 34 * Ingestion rate 0 : cytochrome c reduction 51* 80* Iodination (Iodide) 20 M M Chemotaxis 75*

1

22

Controls

23

5.2 90

18.1** 95

51* 11.4 4.2

88 11.3 3.1* 2.8* 13.8

1.1*

11.0 53

110

Mean f 1 SD

No.

7.0 f 2.0 87 f 7

25 30

76t 16.0 f 6.2 t 7.0 5 16.3 f

8 2.5 1.1 1.3 3.2

30 40 30 30 40

96

32

15 25 30 40

f

-

-

-

-

-

-

7.0 f 2.0 7.0 f 1.3 16.3 f 3.2

0.83* 200

1.03 212

1.61 t 0.29 150 to 2501

33

25 * 85 57*

-

100 f 12 loo* 8 lOOr 7 100 f 11

20 23 22 26

65* 86 -

(1-8) Same legends as under Table 1. (9) Same legends as under Table 3. (10) Same legends as under Table 4. t Control range.

576

EVARIST FELIU e t al.

PMN are tested in control serum. In addition, the deficiency is observed when control PMN are studied in the patient’s serum. This was observed in all cytochrome c reduction deficiencies. However, cytochrome c reduction of PMN from patients 4 and 5 were withm the control range when measured in autologous serum while the serum of these two patients decreased cytochrome c reduction of control PMN. The explanation of this discrepancy might be that the PMN of both these patients showed a very high ingestion rate which should have been accompanied by a vigorous metabolic response [39, 401. This, in fact, was the case except for 0 ; production measured by cytochrome c, reduction (Table 1). The above triad was also observed in all but one (patient 2 1) iodination deficiencies recorded. The massive decrease of MPO-score in PMN from patient 21 might explain the low level of iodination in control serum [ 18, 24, 411. However, the serum of this patient shared with MPO the responsibility for the decreased iodination observed in autologous serum, owing to the improvement of iodination in control serum and inhibition of iodination in control PMN when measured in the patient’s serum. The above triad was again noted in the ingestion rate deficiencies. Thus the serum of patients with cirrhosis diminished PMN cytochrome c reduction (twenty out of twenty-three patients studied), PMN iodination (eight out of twenty-three patients studied) and PMN ingestion rate (three out of twenty-three patients studied). The serum-induced (patients 19,20 and 21) ingestion defect was, as expected, associated with a subsequent deficiency of the oxygen-dependent metabolic events. The ingestion deficiency was not, however, observed with the histochemical test of latex engulfment. This suggests the possibility of a defect in serum opsonin since it is well known that latex particles do not require opsonization prior to ingestion by PMN whereas Klebsiella do. Immunoglobulin or complement levels were not, however, correlated with the ingestion rate. Bailey et al. [42] found in the serum of patients with fulminant hepatic failure a factor which inhibited hexose monophosphate shunt activity of the PMN. The possible relation existing between the factor isolated by these authors and the inhibiting effect of the serum of cirrhotic patients needs further studies. PMN of patients with liver disease also showed serum independent abnormalities (i.e. not induced by the patients’ serum or corrected by control serum in v i m ) . One third of the patients had a decrease MPO-score of their PMN. The slight decrease of MPO observed in the PMN did not induce an iodination deficiency, since these PMN iodinated normally when incubated in control serum. When the decrease in MPO-score was important, as observed in patient 21, it, however, accounted for an iodination deficiency. The PMN of three patients (patients 18, 19 and 22) were deficient in NBT reduction measured with the histochemical test as well as with the quantitative test which was performed in the absence of serum. In the five patients (patients 3, 4, 5 , 12 and 23) with PMN which displayed a hgh ingestion rate of Klebsiella (Table 2) when measured in autologous serum, the ab-

normality was probably serum-independent, since the serum of these patients did not enhance the ingestion rate of control PMN (Table 4). Thus serum-independent PMN abnormalities occur in liver disease but they are less frequent than serum-dependent abnormalities. The abnormalities observed in the PMN of patient 18 are so complex, combining serum-dependent and serumindependent deficiencies, that interpretation is impossible. Biological heterogeneity is often observed in cirrhotic patients and our study on the function of their PMN is a further example of this. Chemotaxis measurements were performed in this study using a different technique to that used previously [7, 20, 211. Cell-directed movement was measured on agarose [36, 371 and not with the Boyden chamber technique [43]. Our results with an attractant different from those previously used [7, 20, 211, confirmed that the serum from cirrhotic patients reduced PMN chemotaxis. The purpose of our measurements of chemotaxis was to check its correlation with other serum-induced PMN abnormalities; no significant correlation was found. In conclusion, the serum in alcoholic cirrhosis and probably in cryptogenic cirrhosis induced deficiencies of PMN function. The most frequently observed were defective chemotaxis, defective 0 ; production measured by cytochrome c reduction, and defective iodination. Defective ingestion and oxygen-dependent metabolic events were less frequently observed. Serum-independent PMN abnormalities were also found in addition to the seruminduced abnormalities in some patients. The molecular mechanism of these abnormalities is not clearly understood, but PMN abnormalities in cirrhosis, may, in part, explain the increased susceptibility t o infection of patients with this disease. References 1 Ratnoff O.D. & Patek A.J., Jr (1942) The natural history of Laennec’s cirrhosis of the liver. Medicine 21, 207-268. 2 Whipple R.L. & Harris J.F. (1950) E. coli septicemia in Laennec’s cirrhosis of the liver. Ann Intern Med 33,462-466. 3 Tisdale W.A. (1961) Spontaneous colon bacillus bacteremia in Laennec’s cirrhosis. Gastroenterology 40, 141-148. 4 Martin W.J.,McHenry M.C., Wellman W.E. & Baggenstoss A.H. (1962) Severe liver disease complicated by bacteremia. Arch Intern Med 109, 555-562. 5 Jones E.A., Crowley N. & Sherlock S. (1967) Bacteremia in association with hepatocellular and hepatobiliary disease. Postgrad Med 43, 7- 1 1 (March Suppl.). 6 Wilson I.D., Onstad G.R., Williams R.C., J r & Carrey J.B., Jr (1968) Selective immunoglobulin A deficiency in two patients with alcoholic cirrhosis. Gastroenterology 54, 253-259. 7 DeMeo A.N. & Andersen B.R. (1972) Defective chemotaxis associated with a serum inhibitor in cirrhotic patients. N Engl J M e d 266,735-740. 8 Bjorneboe M., Jensen K.B., Scheibel I., Thomsen A.C. & Bentzon M.W. (1970) Tetanus antitoxin production and gamma-globulin levels in patients with cirrhosis of the liver. Acta Med Scand 188,541-546. 9 Tomasi T.B., Jr & Tisdale W.A. (1964) Serum gamma-globulin in acute and chronic liver diseases.Nuture 201, 834-835. 10 Wilson I.D., Onstad G. & Williams R.C., Jr (1969) Serum immunoglobulin concentrations in patients with alcoholic liver disease. Gastroenterology 51, 59-67. 11 Thompson R.A., Carter R., Stones R.P., Geddes A.M. & Goodall J.A.D. (1 973) Serum immunoglobulins, complement

POLYMORPHONUCLEAR DYSFUNCTION IN CIRRHOSIS component levels and autoantibodies in liver disease. Clin Exp Immunol 14,335-346. 12 Asherson G.L. (1960) Serum complement levels in systemic lupus erythematosus and other diseases. Aust Ann Med 9, 57-63. 13 Rosen F.S. (1971) The complement system and increased susceptibility to infection. Semin Hematol8, 221 226. 14 Stossel T.P. (1974) Phagocytosis (3 parts). NEngl JMed 290, 717-723,774-780,833-839. 15 Baehner R.L. (1975) Microbe ingestion and killing by neutrophils: normal mechanisms and abnormalities. Clin Haematol 4,609-633. 16 Gallin J.I. & Wolff S.M. (1975) Leucocyte chemotaxis: physiological considerations and abnormalities. Clin Haematol 4,567-607. 17 Stossel T.P. (1975) Phagocytosis: recognition and ingestion. Semin Hematol 12, 83-116. 18 Klebanoff S.J. (197 1) Intraleukocytic microbicidal defects. Annu Rev Med 22,39-62. 19 Klebanoff S.J. (1975) Antimicrobial mechanisms in neutrophilic polynuclear leukocytes. Semin Hematol 12, 117- 142. 20 VanEppsD.E., Strickland R.J. & Williams R.C. (1975) Inhibitors of leukocyte chemotaxis in alcoholic liver disease. A m J Med 59,200-207. 21 Maderazo E.G., Ward P.A. & Quintiliani R. (1975) Defective regulation of chemotaxis in cirrhosis. J Lab Clin Med 85, 621 -630. 22 Senn H.J. & Jungi W.F. (1975) Neutrophil migration in health and disease. Semin Hematoll2, 27-45. 23 Kabat E.A. & Mayel M.M. (1961) Experimental Immunochemistry, 2nd edn, p. 133. C. C. Thomas, Springfield, Illinois. 24 Hakim J., Cramer E., Boivin P., Troube H. & Boucherot J. (1975) Quantitative iodination of human blood polymorphonuclear leukocytes. Eur J Clin Invest 5 , 215-219. 25 Cramer E., Auclair C., Hakim J., Feliu E., Boucherot J., Troube H., Bernard J.F., Bergogne E. & Boivin P. (1977) Metabolic activity of phagocytosing granulocytes in chronic granulocytic leukemia. Ultrastructural observation of a degranulation defect. Blood 50, 93-106. 26 Boyum A. (1968) A one stage procedure for isolation of granulocytes and lymphocytes from human blood. Scand J Clin Lab Invest 21, (Suppl. 97), 51-76. 27 Boyum A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugations and sedimentation at 1 g. Scand J Clin Lab Invest 21, (SUPPI.97), 77-89. 28 Boyum A. (1968) Isolation of leucocytes from human blood further observations. Methylcellulose, dextran and ficoll as

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