IS FATAL PANCREATITIS A CONSEQUENCE OF EXCESSIVE LEUKOCYTE STIMULATION? THE ROLE OF TUMOR NECROSIS FACTOR a Rosamonde E. Banks,*

S.W. Evans, D. Alexander,’ J.T. Whither

M.J. McMahon,’

This study examines the possible role of excessive leukocyte stimulation in the pathogenesis of acute pancreatitis. Levels of tumor necrosis factor (Y (TNF-(u) and elastase-ol, proteinase inhibitor (E-alPI) have been measured as markers of macrophage and neutrophil activation, respectively, in serial plasma samples from 27 patients with acute pancreatitis. Levels of TNF-a did not support the possibility of excessive macrophage activation, but raised levels of E-(u,PI in patients with either the mild or severe form of the illness indicated the activation of neutrophils. Whether this is a consequence of the illness or is a contributory factor in the pathogenesis is not clear. Copyright o 1991 by W.B. Saunders Company

Acute pancreatitis varies in severity from a relatively benign illness with low mortality and few complications to a severe form accompanied by pancreatic necrosis or sepsis, which frequently results in death, usually from multiorgan failure. The overall mortality is approximately 10%’ and is generally considered to result from damage to the pancreas and other organs by circulating pancreatic enzymes. Recently it has been proposed that enzymeinduced damage may be only a contributory factor and that the complex processes underlying the high mortality in severe pancreatitis result primarily from excessive stimulation of leukocytes as a result of severe or persistent pancreatic trauma.* It is hypothesized that the release of lysosomal enzymes and reactive oxygen metabolites following the prolonged stimulus would thus produce further injury, increasing the inflammation and initiating a vicious cycle.* Formation of large amounts of protease-protease inhibitor complexes and cellular debris may exceed the phagocytic capacity of macrophages and result in excess secretion of the polypeptide cachectin (tumor necrosis factor (Y,TNF-ol) into the circulation. Although lesser amounts of TNF may produce predominantly beneficial effects in en-

Department of Chemical Pathology, The Old Medical School, University of Leeds, Leeds LS2 9JT, UK. ‘The University Department of Surgery, The General Infirmary at Leeds, Leeds LS13EX, UK. *Author to whom correspondence should be addressed. Copyright o 1991 by W.B. Saunders Company 1043.4666/91/0301-0006$05.00/O KEY WORDS: pancreatitisiTNF

12

alpha,proteinase

inhibitorielastaselleukocytesl

hancing host defense, prolonged excessive secretion is deleterious and can result in lethal tissue injury similar to that seen in endotoxin shock (reviewed in reference 3). Elastase, a neutrophil lysosomal enzyme released on cell death or during phagocytosis, is rapidly complexed in the circulation by cyl proteinase inhibitor (a,-antitrypsin, o,PI) and, to a lesser extent, ol,-macroglobulin.4 The complex with (Y,PI (E-a,PI) has been shown to be elevated in various inflammatory conditions such as rheumatoid arthritis,’ septicaemia,6.7 ulcerative colitis, and Crohn’s diseaseEs9and recently has been found to be an early marker for the severity of acute pancreatitis.” In this study we have investigated the involvement of both neutrophils and macrophages in acute pancreatitis by measuring levels of E-(w,PI and TNF-a in serial plasma samples from 27 patients with acute pancreatitis.

RESULTS The clinical features of the patients are shown in Table 1. The median daily plasma levels of E-o,PI and TNF-ol and the total leukocyte count for the two groups of severity are shown in Figures 1, 2, and 3, respectively. Sample collection was incomplete for some patients due to factors such as death or discharge prior to day 8. The normal levels of E-o,PI indicated are those previously determined in this laboratory,5 while the normal TNF-(w values were derived from a group of 14 women and 5 men with an age range from 22 to 79 years and have a median value of 2.6 pg/ml. These TNF-a values are lower than those quoted in the literature supplied with the TNF-a kit, where normal CYTOKJNE,

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Table 1. The clinical features of 27 patients with acute pancreatitis included in the study. Grade Mild

Severe

Number

Age (mean)

Sex

19

30-83 years

(61)

14M:5F

8

42-96 years

(67)

4M:4F

levels of 36 controls have a mean value of 6.3 pg/ml and a much wider range. The median levels of E-ol,PI were higher in the “severe” than the “mild” group on days 1 to 8, and significantly so (p < 0.05) on days 2,3, and 5, although there was considerable overlap between the two groups. For the two patients for whom data was available on the day they died, E-a,PI levels were elevated (532 and 860 l~,g/L), but in both cases were declining. The highest level reached was actually in a patient in the “mild” group who had a level of 5,850 &L on admission, which returned to normal by day 4. The majority of patients in both groups for whom data were available had levels that remained elevated throughout the g-day period, although they declined with time. However, 37% of the patients in the “mild” group were discharged before 8 days and omission from analysis of possibly normalizing levels in these patients may have resulted in the slight increase in median levels seen after day 5. No significant differences were seen between the two groups with regard to the levels of TNF-ol although the “severe” group had a higher median level. One

Etiology

Complications

Gallstones (10) Alcohol (1) Post-ERCP (1) Idiopathic (7) Gallstones (3) Alcohol (3) Idiopathic (2)

Pre-existing chest Minor renal (1)

Death (4) Respiratory Pseudocyst

problems

(2) (2)

patient in the “severe” group had a level of 314.8 pg/ml on day 1, which had become normal by day 4 (day 2 and 3 data not available). Many of the patients had levels that remained within normal limits throughout the study or were only slightly elevated. Of the two patients who died and for whom data is available on the day of death, both had slightly elevated levels, one at 22 pg/ml and one at 62.1 pg/ml. The total leukocyte count was not significantly different between the two groups although it was generally higher in the “severe” group. Data was available for only one patient on the day of death who had a greatly elevated count of 17.8 x lo9 cells/L. No significant correlation was seen between white cell count and either E-qP1 or TNF-or levels when the data from both groups and all days was analyzed. A significant correlation between E-ar,PI and TNF-IX was seen (r = 0.415, p = 0.005), however, this was largely influenced by the very high value of 314.8 pg/ml TNF-(w on day 1 for one of the patients with a value of 3,600 ug/L for E-a,PI and was not significant if this point was omitted.

3000 1

l

l

2500

I 3 . 0) -2

2000-

a

1500.

6 31 x--L 51

tTT ti.l lOOOFigure 1. Plasma elastase-a, proteinase inhibitor levels in serial samples from 8 patients with “severe” acute pancreatitis and 19 patients with “mild” acute pancreatitis. Results are ures denote Day 1 = day indicates the

the median the number of admission upper limit

? interquartile range. Figof patient samples per day. to hospital. *, p < 0.05. ---of normal levels.

(1)

* Severe

500 I 01 12

3

4

5 Day

6

7

6

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35-

30-

Severe b Mild Figure 2. Plasma TNF-CY levels in serial samples from 8 patients with “severe” acute pancreatitis and 19 patients with “mild” acute pancreatitis.

1

2

3

4

5

6

Results are the median k interquartile range. Figures denote the number of patient samples per day. Day 1 = day of admission to hospital. ---- indicates the upper limit of normal levels.

7

Day

DISCUSSION In this paper we have examined the hypothesis proposed by Rinderknecht’ that excessive leukocyte stimulation is responsible for the multiple organ failure associated with severe acute pancreatitis. The data suggest that excessive macrophage stimulation is not a persisting phenomenon during severe acute pancreatitis but prolonged stimulation of neutrophils does occur. The pleiotropic macrophage-derived cytokine TNF-(11 mediates a number of events associated with inflammation and the acute phase response, including fever,” prostaglandin synthesis,12 acute phase protein production (reviewed in reference 13), and neutrophil activation.14 In 43 critically ill patients with sepsis, 25% exhibited elevated TNF-cx levels (10 to 100 pg/ml) and the mortality of these patients was twice that for comparable patients who did not exhibit comparable rises in TNF.” In a similar study with 27 patients suffering from septic shock, TNF-a serum levels ranging from 100 to 5,000 pg/ml have been reported.16 However the normal controls with which the septic patients were compared were also found to have a mean level of 75 pg/ml, which is higher than usually reported. It has also been demonstrated that injecting TNF-cx into animals at concentrations associated with endotoxic shock leads to multiple organ failure” and that this is abrogated by administration of monoclonal antibodies to TNF-cL’~ It is conceivable, therefore, that

20. 1816-

2. 0’

1

2

3

4

5

Day Figure 3. Total white cell counts in serial samples from 8 patients with “severe” acute pancreatitis and 19 patients with “mild” acute pancreatitis.

Results are the median f interquartile admission to hospital.

range. Day 1 = day of

Pancreatitisand leukocytestimulation /

TNF-a may contribute to the multiple organ failure associated with severe forms of sepsis, including acute pancreatitis. However, the levels of TNF-a that were measured in the plasma of patients with acute pancreatitis in this study were, in the majority of cases, lower than those detected in the plasma or serum of individuals suffering from septic shock1s,‘” or, indeed, in those detected in normal individuals who had been injected with endotoxin.19 Since the TNF-a levels required to induce multiple organ failure in rodents are similar to the levels measured in patients with endotoxic shock but considerably higher than those we found in pancreatitis, it is unlikely that TNF-(w itself is directly responsible for multiple organ failure in severe acute pancreatitis. One consequence of neutrophil activation is the release of neutrophil elastase. Free elastase is rapidly complexed with 0l,P1.~At the onset (within 24 h) of an attack of acute pancreatitis, E-CX,PIlevels in the severe and mild groups were considerably raised, although the values recorded in the two groups were not significantly different. It is interesting that, on the subsequent four days, levels of E-(Y,PI remained high in the severe group but fell towards the normal range in the mild group. The differences on three of these days were statistically significant. After day 5 there was no significant difference between the two groups and levels of complex continued to return towards the normal range. These results suggest that in severe acute pancreatitis there is a more prolonged period of excessive neutrophil activation than in the majority of mild cases, although there is considerable overlap between the two groups. Even in the two individuals who suffered a fatal attack and for whom data was available at the time of death the levels of E-(w,PI were declining although still greatly elevated. Although this may reflect neutrophil exhaustion, it suggests that if neutrophil activation is a determinant of the severity of acute pancreatitis, the critical event occurs several days prior to death. It is of course possible that prolonged neutrophil activation does not contribute significantly to the severity of the attack but is simply a consequence of it. As a result of increased neutrophil activation, free radical formation might lead to cellular damage either directly or indirectly via total or partial inactivation of proteinase inhibitors,20*2’ thus reducing the inhibition of released pancreatic and neutrophil enzymes. Since we commenced this study, elevated E-cx,PI levels have been reported in a large series of patients with acute pancreatitis, with similar trends and levels to those reported here although expressed as mean levels rather than median.” Significant differences were found, however, between day 1 levels in their mild group versus severe and lethal groups, which we did not find. This is probably a reflection of the larger size of their groups, particularly those groups of patients

15

with more severe illness. They also found that E-a,PI was an earlier marker than C-reactive protein (CRP) for severity. Although the acute phase protein data has been determined in our study together with levels of complexed cY,-macroglobulin” and the trends were similar to those reported by Gross et al.,” our study is too small to examine the relative predictive values of these proteins. The data presented in this paper suggest that excessive neutrophil activation occurs in severe acute pancreatitis although macrophage activation as evidenced by plasma levels of TNF-IX does not seem to be pronounced. The trigger for neutrophil activation and the reason for the more prolonged elevation of E-a,PI levels in the severe cases have still to be determined and may not be just a consequence of increased production, but may also reflect impaired clearance of proteinase-inhibitor complexes by macrophages. An impaired phagocytosis of zymosan particles by macrophages from patients with pancreatitis has been observed which appeared to be specific to pancreatitis and to persisit for up to 8 days after admission (M.J. McMahon, unpublished observations). Its cause is unknown but the present study suggests it is not a consequence of excessive macrophage stimulation.

MATERIALS AND METHODS A diagnosis of acute pancreatitis was made in the 27 patients included in this study on the basis of a consistent clinical picture including a plasma amylase level exceeding 1,200 IU/L (“Phadebas,” Pharmacia, Milton Keynes, UK). The etiology of the attack was classified as: (i) biliary, when gallstones were demonstrated by ultrasound, ERCP, laparotomy, or autopsy; (ii) alcohol, when average daily consumption exceeded 50 g in the absence of other causes; (iii) other, when an alternative cause was identified; and (iv) idiopathic, when no cause was identified. The severity of the attacks was graded on the basis of outcome, with a “mild” attack being one in which only minor complications occurred and a “severe” attack being characterized by death, major organ failure, or pancreatic pseudocyst, abscess,or necrosis.

Citrated blood samples were collected from the patients on the day of admission to hospital (day l), usually within 24 h of the onset of symptoms. Samples were collected daily until day 8 or until discharge from hospital if sooner. Plasma was obtained by centrifugation of the blood samples at 1,500g for 10 min and stored frozen at -40°C until analysis. EDTA anticoagulated blood samples were also obtained, and a full blood count was performed.

E-a,PI was measured as previously described’ using an ELISA method. The sheep anti-elastase antibody used in this assaywas obtained from the same source (Serotec, Oxford, UK) as that previously used in a similar assaywhere it was shown to be specific for neutrophil elastase and not to cross-react with pancreatic e1astase.23TNF-a levels were determined using a commercially available immunoradiomet-

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ric assay (Medgenix Diagnostics, Belgium) according to the manufacturers instructions. Statistical analysis was performed by the two-tailed Mann-Whitney U test.

Acknowledgment We are grateful to Bayer UK for the support of David Alexander. REFERENCES 1. Mayer AD, Airey M, Hodgson J, McMahon MJ (1985) Enzyme transfer from pancreas to plasma during acute pancreatitis. The contribution of ascitic fluid and lymphatic drainage of the pancreas. Gut 26:876-881. 2. Rinderknecht H (1988) Fatal pancreatitis, a consequence of excessive leukocyte stimulation? Int J Pancreatol3:105-112. 3. Tracey KJ, Vlassara H, Cerami A (1989) Cachectin/tumour necrosis factor. Lancet i:1122-1126. 4. Ohlsson K, Olsson I (1974) Neutral proteases of human granulocytes. III. Interaction between human granulocyte elastase and plasma protease inhibitors. Stand J Clin Lab Invest 34:349-355. 5. Banks RE, Evans SW, Taylor KF, Bird HA, Whither JT (1990) Measurement of plasma concentrations of ploymorphonuclear elastase-a, proteinase inhibitor (elastase-cu, antitrypsin) in patients with rheumatoid arthritis: interference by rheumatoid factor. Ann Rheum Dis 49:18-21. 6. Speer CP, Rethwilm M, Gahr M (1987) Elastase-cy,proteinase inhibitor: an early indicator of septicaemia and bacterial meningitis in children. J Paediatr 111:667-671. 7. Duswald K-H, Jochum M, Schramm W, Fritz H (1985) Released granulocytic elastase: an indicator of pathobiochemical alterations in septicaemia after abdominal surgery. Surgery 98:892899.

8. Adeyemi EO, Neumann S, Chadwick VS, Hodgson HJF, Pepys MB (1985) Circulating human leucocyte elastase in patients with inflammatory bowel disease. Gut 26:1306-1311. 9. Fink PC, Suin De Boutemard C, Haeckel R, Wellmann W (1988) Endotoxaemia in patients with Crohn’s disease: a longitudinal study of elastas&-proteinase inhibitor and Limulus-amoebocytelysate reactivity. J Clin Chem Clin Biochem 26:117-122. 10. Gross V, Scholmerich J, Leser H-G, Salm R, Lausen M, Ruckauer K, Schoffel U, Lay L, Heinisch A, Farthmann EH, Gerok W (1990) Granulocyte elastase in assessment of severity of acute pancreatitis. Comparison with acute phase proteins C-reactive protein, cy,-antitrypsin, and protease inhibitor u,-macroglobulin. Dig Dis Sci 35:97-105. 11. Dinarello CA, Cannon JG, Wolffson JG, Bernheim HA,

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Beutler B, Cerami A, Figari IS, Palladino MA, O’Connor JV (1986) Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 1. J Exp Med 163:1433-1450. 12. Nakamura H, Seto Y, Motoyoshi S, Kadokawa T, Sunahara N (1988) Recombinant human tumor necrosis factor causes longlasting and prostaglandin-mediated fever, with little tolerance in rabbits. J Pharmacol Exp Ther 245:336-341. 13. Whither JT, Thompson D, Billingham MET, Kitchen EA (1989) Acute phase proteins. In Chang J, Lewis AJ (eds) Pharmacological Methods in the Control of Inflammation, Alan R Liss Inc, London, pp 101-128. 14. Nathan CF (1987) Neutrophil activation on biological surfaces: massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes. J Clin Invest 80:15501560. 15. Debets JMH, Kampmeijer R, Van Der Linden MPMH, Buurman WA, Van Der Linden CJ (1989) Plasma tumor necrosis factor and mortality in critically ill septic patients. Crit Care Med 17:489-494. 16. Damas P, Reuter A, Gysen P, Demonty J, Lamy M, Franchimont P (1989) Tumor necrosis factor and interleukin-1 serum levels during severe sepsis in humans. Crit Care Med 17:975-978. 17. Tracey KJ, Beutler B, Lowry SF, Merryweather J, Wolpe S, Milsark IW, Hariri RJ, Fahey TJ, Zentella A, Albert JD, Shires GT, Cerami A (1986) Shock and tissue injury induced by recombinant human cachectin. Science 234:470-474. 18. Tracey KJ, Fong Y, Hesse DG, Manogue KR, Lee AT, Kuo GC, Lowry SF, Cerami A (1987) Anti cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature 330:662-664.

19. Michie HR, Manogue KR, Spriggs DR, Revhaug A, O’Dwyer S, Dinarello CA, Cerami A, Wolff SM, Wilmore DW (1988) Detection of circulating tumor necrosis factor after endotoxin administration. New Engl J Med 318:1481-1485. 20. Dean RT, Nick HP, Schnebli HP (1989) Free radicals inactivate human neutrophil elastase and its inhibitors with comparable efficiency. Biochem Biophys Res Commun 159:821-827. 21. Padrines M, Schneider-Pozzer M, Bieth JG (1989) Inhibition of neutrophil elastase by alpha-1-proteinase inhibitor oxidized by activated neutrophils. Am Rev Respir Dis 139:783-790. 22. Banks RE, Evans SW, Alexander D, Van Leuven F, Whither JT, McMahon MJ (in press) Alpha,-macroglobulin status in acute pancreatitis. Elevated levels of a,-macroglobulin-protease complexes in severe and mild attacks. Gut. 23. Boger C, Yuan H-Z, Schultek T, Tegtmeier K-F, Wood WG (1988) Development and clinical evaluation of immunoluminometric assays for lactoferrin and elastase-a,-proteinase inhibitor complexes in body fluids with special references to brochoalveolar lavage and neonatal sepsis. J Clin Chem Clin Biochem 26:645-651.

Is fatal pancreatitis a consequence of excessive leukocyte stimulation? The role of tumor necrosis factor alpha.

This study examines the possible role of excessive leukocyte stimulation in the pathogenesis of acute pancreatitis. Levels of tumor necrosis factor al...
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