Enhanced granuloma angiotensin I converting enzyme activity associated with modulation in murine schistosomiasis

GASTROENTEROLOGY

1981;81:48-53

Enhanced Granuloma Angiotensin I Converting Enzyme Activity Associated with Modulation in Murine Schistosomiasis JOEL V. WEINSTOCK, J. BERNARD GEE

DOV L. BOROS, and

Department of Medicine, Division of Gastroenterology, and Department of Immunology and Microbiology, Wayne State University, Detroit, Michigan; and Department of Medicine, Pulmonary Division, Yale University, New Haven, Connecticut

Angiotensin-converting enzyme activity was detected in serum and in isolated granuiomas from liver, colon, and ileum of CBA/J mice infected with schistosomiasis. Murine schistosomiasis mansoni is a cell-mediated granulomatous disease associated with a spontaneous decrease in the size of the granulomatous inflammation at the chronic stage of the infection (modulation). Associated with modulation of the granulomatous response, enhanced angiotensin-converting enzyme activity was measured in serum and in isolated liver and colon granulomas. Ileal granulomas, which do not modulate size, did not show a change in this activity. Isolated synchronous lung granulomas, induced by the pulmonary embolization of schistosome eggs into normal and sensitized mice, demonstrated increasing angiotensin-converting enzyme activity during involution. Prior sensitization resulted in enhanced granuloma angiotensin-converting enzyme activity and a peak serum angiotensin-converting enzyme activity corresponding with the maximal size of the inflammatory response. Angiotensin-converting enzyme activity was associated with the granuloma macrophage/epithelioid cell which secreted angiotensin-converting enzymes when cultured in vitro. Granuloma cells depleted of macrophages had no angiotensin-converting enzymes activity. These studies suggest that angiotensin-converting enzymes activity in schistosome egg-induced Received June 25, 1980. Accepted January 27, 1981. Address requests for reprints to: Joel V. Weinstock, M.D., ASsistant Professor of Medicine, Chief, Section of Gastroenterology, Hutzel Hospital Medical Unit, 4707 St. Antoine, Detroit, Michigan 48201. This work was supported by NIH research grant AI-12913 and RR-05384. 0 1981 by the American Gastroenterological Association 0018-5085/81/070048-06$02.50

granulomas is associated with the macrophage and increases with modulation and involution of the granulomatous response. This enzyme may participate in regulation of this inflammatory lesion. Murine schistosomiasis appears to be a good model to study the relationship of angiotensin-converting enzyme activity to granulomatous inflammation.

Increased serum angiotensin I converting enzyme (ACE) (Kininase II) activity has been detected in some patients with sarcoidosis and occasionally in patients with other granulomatous conditions (l-5). Angiotensin-converting enzyme activity is also high in granulomatous lymph nodes from patients with sarcoidosis (2,6). Furthermore, ACE is present in epithelioid cells of the sarcoid granuloma (7), in pulmonary alveolar macrophages from cigarette smokers (a), and in Gaucher and endothelial cells (912). The significance of enhanced ACE activity associated with some granulomatous diseases is currently unknown. In preliminary experiments, we observed high ACE activity in granulomas induced by Schistosoma mansoni eggs (13). Murine schistosomiasis mansoni, a granulomatous disease of cell-mediated etiology, is associated with a spontaneous decrease in the size of the granulomatous inflammation at the chronic stage of infection (modulation) (14). This prompted us to search for an association between diminished granuloma size and enhanced granuloma and serum ACE activity. Sequential determinations of ACE activity in aging synchronous pulmonary granulomas (involution) were also made. In the present communication, we report that granuloma ACE activity is enhanced by both modulation and involution of granulomatous inflamma-

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ACE ACTIVITY ASSOCIATED WITH MODULATION OF GRANULOMATOUS INFLAMMATION

tion, and that the probable source of ACE activity in isolated liver granulomas is the macrophage.

Methods Animals

Used and Method of Infection

Female CBA/J mice (Jackson Memorial Laboratories, Bar Harbor, Me.) 6-7 wk of age were infected by subcutaneous injection of 25 cercariae of the Puerto Rican strain of Schistosoma mansoni. Perfusion of the portal vein routinely yielded 8-10 worm pairs.

Jsolation of Granulomas Granulomas were isolated from various organs by modification of the method of Pellegrino and Brener (15). Subsequently, the isolated granulomas were washed three times with Hank’s balanced salt solution (HBSS), then packed at 300 g to remove supernatant. Lung and small bowel granulomas were isolated by a similar grinding procedure, and the resulting tissue suspensions were collected on No. 80 stainless steel screens and washed repeatedly with HBSS. The small bowel was irrigated clean with HBSS before mincing.

Isolation (16) and Culture of Granuloma

Cells

Isolated liver granulomas from mice infected for 8 wk were placed in a 50-ml siliconized Erlenmeyer flask with RPMI-1640 medium (RPMI) (GIBCO, Grand Island Biological Co., Grand Island, N.Y.), 1 ml/O.5 ml loosely packed tissue volume, containing 10% fetal calf serum (heat inactivated at 56°C for 30 min) and collagenase, 1600 U/ml (both from Sigma Chemical Co., St. Louis, MO.). The tissue suspension was agitated at 37’C in a shaker water bath for 50 min. The mixture was then centrifuged at 300 g for 5 min to remove the collagenase solution and was washed with RPMI. The loosened granulomas were dispersed by gently teasing and pressing them through a No. 80 stainless steel screen into a siliconized, 300-ml beaker. The dispersed cells were washed three times at 4’C with RPM1 in siliconized, 16 x 125-mm glass test tubes. The yield was approximately 3 x lo7 cells/liver with 00% viability as determined by trypan blue exclusion. They were then resuspended in 20 ml RPM1 containing 10% fetal calf serum, 2.0 mM/ml glutamine, 100 U/ml penicillin, and 100 pg/ml streptomycin (all from GIBCO) and cultured on 5cm glass tissue culture dishes at 37’C in a 5% CO, atmosphere for 2 h. The nonadherent cells were subsequently removed by washing the cell monolayers three times with RPM1 medium. The nonadherent fraction was incubated for 1 h at 37’C with carbonyl iron powder (Pfaltz and Bauer, Inc., Stamford, Conn.) (1 X 10’ cells/100 mg iron) and passed through a magnetic field to remove phagocytic cells. The resulting nonadherent, nonphagocytic fraction [<5% phagocytized neutral red dye (l7)] containing about 3% macrophagelike cells, 80% eosinophils, and 17% lymphocytes was placed in 0.5 ml distilled water, sonicated 15 s

49

on ice, and assayed for ACE activity. Some nonadherent cells were maintained in culture for 24 h, and ACE activity of medium and cells was determined. The adherent cell population was >95% macrophagelike by morphologic criteria. These cells were lo-30 pm in diameter with oval eccentric nuclei. They displayed numerous vacuoles and pseudopodia. Some multinucleated giant cells were seen. Ninety-five percent of these adherent cells phagocytized >lO latex beads, and 80% were nonspecific esterase positive (18). The adherent cells were maintained for 24 h in RPM1 medium supplemented with fetal calf serum, glutamine, penicillin, and streptomycin as previously described. Culture medium was collected, and cell monolayers were washed three times with RPMI. The adherent cells were then removed with a rubber policeman and 0.5 ml distilled water. They were subsequently sonicated for 15 s on ice, and both the lysed cells and culture medium were assayed for ACE activity.

Isolation of Schistosome Eggs and Production of Soluble Egg Antigen (SEA) With a modification of the method of Coker and von Lichtenberg (IO), schistosome eggs were isolated from the livers of mice infected for 8 wk with 200 cercariae of Schistosoma mansoni. Soluble egg antigen was obtained by homogenization of the eggs in an iced Ten Broeck tissue grinder followed by ultracentrifugation at 100,000 g for 2 h (20).

Induction of Synchronous Granulomas (21)

Pulmonary

Eggs were suspended in phosphate-buffered saline at a concentration of 8000 eggs/ml, and 4000 eggs were injected into the tail vein of 8-wk-old normal mice using a tuberculin syringe with a 21-gauge needle. These eggs embolized to the lungs inducing focal granulomas. Another group of normal mice were sensitized intraperitoneally with 1000 eggs 1 wk before intrapulmonary egg challenge.

Assay of ACE Activity, Protein, and DNA Concentrations All samples were stored at -7O’C for future protein and ACE activity determinations for periods up to 2 mo. Under these conditions, there is no change in serum ACE activity (1,22) or granuloma ACE activity. Angiotensin-converting enzyme activity was assayed by a spectrophotometric method (1). The substrate was hippuryl-L-histidyl-L-leucine, an angiotensin I analog. Activity in serum was expressed as hippuric acid (nmol/min)/ml and in tissue and lysed monolayers as hippuric acid (nmol/min)/ mg protein. We also measured ACE activity with p[3H]benzoyl-Phe-Ala-Pro-OH, a carboxy-terminal analog of bradykinin-potentiating peptide 5a, a substrate provided by Dr. J. Ryan (University of Miami) and p[3H]benzoyl-Gly-Gly-OH (Ventrex, Portland, Me.). To determine ACE activity in isolated granulomas, gran-

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WEINSTOCK ET AL.

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Results

ACE Actrdy Granulomo Volume

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ACE Activity in Isolated Granulomas and Sera of Mice Infected with Schistosomiasis

IO WEEKS

1

15

AFTER

20

25

35

INFECTION

Figure 1. Change in A. ACE activity and volume of liver granulomas, and B. serum ACE activity after infection of mice with schistosomiasis. Volume data are mean values of 120observations pooled from four to six livers f SEM. ACE activity data are means from four to nine mice f SEM.

ulomas were ground in a mortar with a pestle for z min (ZOOmg tissue/3 ml HBSS) at O’C, and subsequently sonicated for 30 s. Protein concentration (23) and ACE activity were measured in these granuloma extracts. DNA content of isolated liver granulomas was also determined (24,25).

Determination

of Granuloma

Volume

Granuloma diameter was measured in stained histologic sections of 2 cm of terminal ileum, right half of colon and/or lungs by a person who was unaware of the identity of the sections. Nonconfluent granulomas containing parasite eggs were located microscopically in the liver, lung, or lamina propria. Their diameters were measured with an image-splitting eyepiece (Vickers Instrument, Woburn, Mass.) by determining the mean of two measurements made across perpendicular axes. Granuloma volume was calculated from mean granuloma diameter.

Additional

Controls and Statistical

considered

significant.

ACE Activity in Synchronous Granulomas

Pulmonary

Synchronous pulmonary granulomas were induced in normal and previously sensitized mice to

Methods

Angiotensin-converting enzyme activity was measured in SEA and in sonicated schistosome-egg, suspensions, as well as in normal mouse serum, liver, lung, small bowel, and colon. Data were subject to the Student’s t-test to determine significant differences between groups. p-Values of co.05 were

An increase in ACE activity in isolated liver granulomas relative to granuloma protein content occurred between 6 and 35 wk of infection. This corresponded with a decrease in mean intrahepatic granuloma volume (Figure 1A). Angiotensin-converting enzyme activity also increased relative to granuloma DNA content [6 wk136.6, 20 wk-632.7, hippuric acid (nmoles/min)/mg 2-deoxypentose]. The protein content of isolated liver granulomas from animals infected for 8 wk was slightly higher than that for 20 wk [8 wk-28.7 & 1, 20 wk-20.1 fl mg protein/g granuloma wet wt], whereas DNA content did not change (8 wk-1.14 fO.O3,2O wk-1.11 f 0.4 mg &deoxypentose/g granuloma wet wt). Furthermore, serum ACE activity was increased from 18 to 35 wk to infection (p < 0.01) (Figure 1B). Antiotensin-converting enzyme activity was also measured in isolated colonic and ileal granulomas to determine whether they also contained the enzyme and to see whether changes in ACE activity again corresponded with modulation of granuloma size. In the colon, the size of the granulomatous response peaks at 8 wk of infection and is fully modulated by 20 wk. The size of the ileal response to schistosome eggs, however, is small and does not modulate (manuscript in preparation). ACE activity in isolated coIonic, but not ileal granulomas showed an increase from 8 to 20 wk of infection (p < 0.0005) (Figure 2A and B).

Figure 2. A. ACE activity and B. mean volume of ileal and colon granulomas of mice infected for 8 or 20 wk. ACE activity data are means pooled from six mice f SEM. Volume data are mean values of 120 observations from six mice f SEM.

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July 1981

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ASSOCIATED

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ACE Acttvlty GranulamaVolume

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lations were 80% eosinophils and 17% lymphocytes and demonstrated essentially no ACE activity when cultured for 2 or 24 h. Culture medium from only 24 h adherent cell monolayers demonstrated ACE activity [34.l +7.7 hippuric acid (nmol/min)/mg protein in monolayer, ~frSEMI. -50

-25 ‘: x -0

OJ-----_

“E E

5 75 @

’5o- Unsensltlred

t -50

-25

-0 WEEKS Figure 3. ACE activity

and mean volume of synchronous lung granulomas as compared with the duration of the granulomatous response in sensitized and unsensitized mice. Volume data are mean values of 100 observations pooled from five mice f SEM. ACE activity data are means from nine mice f SEM.

determine the effect of sensitization and granuloma age on granuloma and serum ACE activity. In mice sensitized by schistosome eggs, highest ACE activity was detected in isolated pulmonary granulomas after the peak inflammatory response (Figure 3). Serum ACE activity also rose but was maximal coresponding with maximal granuloma size (Figure 4). In normal unsensitized mice, the granulomatous response was smaller as compared with pulmonary granulomas induced in sensitized animals. Both granuloma and serum ACE activity peaked after the maximal inflammatory response and did not achieve levels as high as those seen in sensitized animals (Figures 3 and 4).

Granuloma ACE as a More Generalized Peptidyldipeptidase To determine whether granuloma homogenates from 8 wk and 20 wk infected mice possessed more generalized dipeptidyl carboxypeptidase activity, we also measured activity with p-[3H]hippuryl-Gly-Gly-OH and a carboxy-terminal analog of bradykinin-potentiating peptide 5a, p-[“HIbenzoyl-Phe-Ala-Pro-OH. These assays detected ACE activity comparable to that detected with hippurylL-histidyl+leucine (Figure 5). Data for p-[“H]benzoyl-Phe-Ala-Pro-OH are not shown. This activity with these substrates was 100% inhibited by lo-"M SQ 14225 and inactivated by heating to 50°C for 30 min. ACE Activity in Schistosome Normal Tissue

Eggs and

In order to eliminate the possibility that the schistosome eggs produced ACE, egg homogenates and SEA were tested and found negative for ACE activity. However, ACE activity was detectable in serum, lung, liver, and intestine of normal mice

Probable Source of ACE Activity Angiotensin-converting enzyme activity was detected in lysed adherent cell monolayers derived from dispersed liver granulomas of 20 wk of infected mice cultured for 2 or 24 h at 37’C [2 h-3.9 20.9, 24h-0.8 fO.l hippuric acid (nmol/min)/mg protein, + SEMI. These cells were >95% phagocytic, 80% nonspecific esterase positive, and had the morphologic appearance of macrophages. Granuloma cells depleted of phagocytic and adherent cell popu-

01

I

2

3

4

WEEKS

Figure 4. Change in serum ACE activity with age of synchronous lung granulomas in sensitized and unsensitized mice. ACE activity data are means from nine mice f SEM.

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WEINSTOCK ET AL.

Figure 5. ACE activity of liver granuloma homogenates from mice infected for 8 or 20 wk measured with the substrate p-[3H]Hippuryl-Gly-Gly-OH. Data are means from six mice f SEM.

[serum-180.8 f8 nmol/min/ml, n = 12; lung-44.7 f7.5 nmol/min/mg protein, n = 8; liver-O.85 f0.2 nmol/min/mg protein, n = 8; small intestine-l.27 f 0.38 nmol/min/mg protein, n = 5 (all SEM)].

Discussion Previous publications have demonstrated that granuloma formation is spontaneously diminished in organs of mice at the chronic phase of the infection with Schistosoma mansoni (modulation) (14,26). Modulation of the granulomatous response may depend in part on the activity of suppressor T lymphocytes (27) present in the spleen or within the lesions (28). In the present paper, we examined another facet of the modulated granulomatous response: that of granuloma-related ACE activity. Sequential measurements of liver granuloma sizes confirmed previous observations which reported a gradual decrease in mean lesion size with increasing duration of infection. Correlated with diminishing lesion volume, granuloma homogenates showed increased ACE activity relative to protein and DNA content. This represented an actual increase in ACE activity since DNA concentration of isolated liver granulomas from 8 and 20 wk infected mice were identical, and protein concentration was only modestly different. An interesting new observation was the comparison of the size of colonic and ileal granulomas at the peak (8 wk) and the modulated (20 wk) phase of the granulomatous response. Whereas lesions in the colon underwent the process of spontaneous modulation with chronic infection, the ileal granulomas, initially smaller than their hepatic or colonic counterparts, did not diminish in size. Concomitant with chronic infection, colonic but not ileal granuloma homogenates had greatly increased ACE activity. As a corollary to the above, a similar relationship between involuting synchronous pulmonary gran-

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ulomas and elevated ACE activity could also be established in unsensitized schistosome egg-injected normal mice. Prior sensitization to schistosome eggs resulted in an enhanced pulmonary granulomatous response and an accelerated involutional phase. The accelerated involution of anamnestic granulomas was accompanied by augmented ACE activity. Modulation and involution of granulomatous inflammation are distinct processes. Modulation occurs in the presence of abundant antigen and involves strong suppressor T-lymphocyte activity. On the other hand, involution refers to a decrease in the intensity of the granulomatous inflammation associated with dissolution and elimination of the antigenie nidus. The factors(s) stimulating high ACE activity during both modulation and involution are unknown. It does appear, however, that ACE activity detectable in granuloma homogenates had the features of peptidyldipeptidase and was therefore probably due to the presence of a true angiotensin-converting enzyme. This was indicated by the capability of granuloma homogenates to split an analog of angiotensin I and other substrates of ACE and by the complete inhibition of this enzyme activity by ACE inhibitor SQ 14225. A close, but not complete, correlation could also be seen between high ACE activity of modulated granulomas and the level of circulating serum ACE activity. This was pronounced in infected mice and in normal animals which carried primary synchronous pulmonary granulomas. These data are consonant with clinical observations which showed increased ACE activity in the circulation and granulomatous lymph nodes of patients with sarcoidosis (l-4,6) and other diseases (5). It is tempting to speculate that the source of the increased ACE activity in the circulation is the tissue granulomas. Fractionation of the dispersed granuloma cells into adherent cells containing macrophages and epithelioid cells vs. lymphocytes and eosinophils showed that the former group was the probable source of the enzyme within the lesions. These data confirm in an animal model the clinical observations which traced ACE activity to epithelioid and giant cells in the sarcoid granuloma (7) and to the bronchoalveolar macrophage (8). The contribution, if any, toward total granuloma ACE activity of endothelial cells (12) from neovascularized granulomas is unknown. The biologic significance of ACE activity in granulomatous inflammatory systems is yet to be determined. This enzyme mediates the conversion of angiotensin I to angiotensin II, which has an important physiologic function in the regulation of blood pressure and volume (29). ACE, which has peptidyldi-

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peptidase activity, also cleaves bradykinin, a vasoactive agent which participates in acute inflammatory responses (30). Previous observations have shown that ellagic acid, which depresses kininogen levels and kinin formation, does slightly suppress the primary schistosome-egg granuloma in the normal mouse lung. Thus, ACE could inactivate bradykinin and serve an antiinflammatory function, However, it is equally plausible that ACE is acting upon some other yet unidentified mediator(s) involved in the inflammatory process. Considering that modulation and involution are somewhat distinct processes as previously discussed, it is perceivable that ACE may have multiple functions within the granuloma. This investigation offers insight into the biologic significance of this enzyme associated with granulomatous inflammation. Future studies are needed to establish the role of ACE in the modulation and involution of the granulomatous response in murine schistosomiasis.

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J. Elevation of serum angiotensin-converting enzyme (ACE) level in sarcoidosis. Am J Med 1975;59:365-72. Silverstein E, Friedland J, Lyons HA, et al. Elevated angiotensin-converting enzyme in granulomatous lymph nodes and serum in sarcoidosis. Clinical and possible pathologic significance. Ann NY Acad Sci 1976;278:498-513. Silverstein E, Friedland J, Kitt M, et al. Increased serum angiotensin-converting enzyme in sarcoidosis. Isr J Med Sci 1977;13:995-lotm. Studdy P, Bird R, Geraint James D, et al. Serum angiotensinconverting enzyme (SACE) in sarcoidosis and other granulomatous diseases. Lancet 1978;ii:1331-4. Gronhagen-Riska C, Kurppa K, Fyhrquist F, et al. Angiotensin-converting enzyme and lysozyme in silicosis and asbestosis. Stand J Respir Dis 1978;59:228-31. Silverstein E, Friedland J, Lyons HA, et al. Markedly elevated angiotensin-converting enzyme in lymph nodes containing non-necrotizing granulomas in sarcoidosis. Proc Nat1 Acad Sci USA 1976;73:2137-41. Silverstein E, Friedland J, Drooker M, et al. Angiotensin converting enzyme from human-lung-purification, preparation of a specific antibody and immunofluorescent localization in sarcoidosis epithelioid and giant-cells. Clin Res 1979;27(2):464. Hinman LM. Stevens C, Matthay RA, et al. Angiotensin convertase activities in human alveolar macrophages: effect of cigarette smoking and sarcoidosis. Science 1979;265:262-3. Lieberman J, Beuther E. Elevation of serum angiotensin-converting enzyme in Gaucher’s disease. N Engl J Med 1976; 294:1442-4. Silverstein E, Friedland J. Elevated serum and spleen angio-

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