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Increased ascitic level of hyaluronan in liver cirrhosis
Increased ascitic level of hyaluronan in liver cirrhosis KAR NENG LAI, CHEUK CHUN SZETO, CHRISTOPHER W. K. LAM, KA BIK LAI, TERESAY. H. WONG, and JOSEPH C. K. LEUNG HONGKONG
Hyaluronan (HA) is a polysaccharide that forms a critical component of extracellular matrixes. It is present in high concentrations in tissues undergoing remodeling and morphogenesis. Serum HA is elevated in patients with chronic liver disease, and this has been considered to be caused by impaired degradation by the liver endothelial cells. We studied the level of HA in the ascitic fluid and plasma from 27 patients with cirrhotic ascites. These values were compared with peritoneal dialysate effluent (PDE) and plasma from 33 patients with uremia who were undergoing continuous ambulatory peritoneal dialysis (CAPD). The median HA levels in ascitic fluid and plasma from our 26 patients with cirrhosis were significantly higher than corresponding PDE and plasma values from the 33 CAPD patients (p < 0.0001). The median peritoneal/plasma ratios of creatinine, albumin, and immunoglobulin G in either cirrhotic or CAPD patients were less than unity. In contrast, the median peritoneal/plasma ratios of HA in both groups of patients exceeded one with a higher peritoneal/plasma ratio of HA in patients with cirrhosis (p = 0.0035). A significant correlation was observed between the ascitic level of HA and interleukin-I~, interleukin-6, or transforming growth factor-~. Our in vitro cell culture studies revealed that HA is synthesized by both mesothelial cells and macrophages. We observed an additive effect in the synthesis of HA by mesothelial cells when the macrophage-conditioned medium was added to the RPMI culture medium. We conclude that a high level of HA is found in ascites from patients with cirrhosis. Our results strongly suggest that simultaneous increased synthesis of HA by the peritoneal cells and a reduction of degradation by liver endothelial cells occur in these patients with cirrhosis with ascites. This event of increased HA synthesis may be contributory to remodeling and regeneration of the peritoneal lining. (J Lab Clin Med 1998; 131:354-9) Abbreviations: bFGF = basic fibroblast growth factor; CAPE) = continuous ambulatory peritoneal dialysis; HA = hyaluronan; IgG = immunoglobulin G; IL = interleukin; PDE = peritoneal dialysate effluent; TGF-~= transforming growth factor-~
H
yaluronan is an unbranched high-molecularweight anionic polysaccharide that forms a critical component of extracellular matrixes. ~ HA can be synthesized by most cells and is present in From the Department of Medicine, Queen Mary Hospital, University of Hong Kong; and the Departments of Medicine and Chemical Pathology, Prince of Wales Hospital, Shatin. Supported by Grant HKU 4210/97M from the Research Grant Council and by the Thomas and Rita Liu Research Fund. Submitted for publication June 17, 1997;revision submitted Nov. 24, 1997; accepted Dec. 4, 1997. Reprint requests: K. N. Lai, Department of Medicine, University of Hong Kong, Room 409, Professorial Block, Queen Mary Hospital, Pokfulam, Hong Kong. Copyright © 1998 by Mosby, Inc. 0022-2143/98 $5.00 + 0 5/1/87994
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high concentrations in tissues undergoing remodeling and morphogenesis, where it acts as a support for cell adhesion and locomotion. It has also been shown to influence cell differentiation, proliferation, and aggregation, and it appears to play a critical role in the early stages of wound healing. 2-4 Some of the H A produced in the peripheral tissues is carried from the tissues by lymph flow to systemic circulation. Most of the HA is degraded in the lymph nodes, but a small percentage reaches the bloodstream, from where it is rapidly taken up by the liver endothelial cells and is degraded. 2 The serum concentration of HA is increased in chronic liver disease and has shown to be a predictor of development of cirrhosis. 5-9 The measurement of serum H A has also been shown to be useful in the monitoring of graft function after liver transplantation. 10,11 Rejection
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episodes h a v e b e e n associated with a clear rise in H A . M e t a b o l i c studies w i t h injected H A have indicated that the liver m a y be a major site for the uptake of circulating H A in h u m a n beings. 12 A n elevated s e r u m H A level in patients with chronic liver diseases is considered to be the sequel of impaired clearance by liver endothelial cells.5,12 In this study w e h a v e e x a m i n e d the ascitic l e v e l o f H A in patients w i t h cirrhosis, and the source o f H A in ascites was explored. METHODS Patients• The study included 27 randomly selected patients with cirrhosis with ascites (20 men and 7 women, aged 57.8 + 14.9 years [mean __ SD]). Cirrhosis was diagnosed either by liver biopsy or clinical features of portal hypertension. The subjects were admitted for symptomatic treatment of ascites with no evidence of bacterial peritonitis. None of these patients had hepatic encephalopathy at the time of our study. The causes of chronic liver disease were chronic hepatitis B virus infection (19), chronic hepatitis C virus infection (6), and alcoholism (2). All but two of these patients with cirrhosis had normal renal function tests. None of these patients had paracentesis or bacterial peritonitis within 4 months of the study period. Thirty-three patients with uremia (20 men and 13 women, aged 55.6 +_ 11.3 years [mean + SD]) undergoing stable CAPD treatment and 26 healthy subjects (17 men and 9 women, aged 52.5 + 14.2 years [mean _+ SD]) served as control subjects. Patients with uremia in whom peritonitis developed within 4 months of the study period were excluded. Informed consent was obtained for collection of ascites and blood samples with approval of our hospital ethical committee. Determination of HA, IL-I [~, IL-6, TGF-[~, and bFGF concentrations in ascitic fluid or PDE. A 50 ml sample of ascitic fluid was collected under aseptic conditions from each cir-
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rhotic patient on admission before any alteration of medication. The ascitic fluid was centrifuged at 1600 rpm for 10 minutes at room temperature. Cell-free ascitic fluid was collected and stored at -70 ° C until analysis for HA and cytokines. A blood sample was taken at the time of ascitic fluid collection for determination of the plasma and ascitic concentrations of creatinine, albumin, IgG, and HA. Ascitic fluid samples were examined to ensure absence of microbes or malignant cells. Therapeutic paracentesis with removal of 1 liter of ascitic fluid was also performed in 12 patients on admission, and peritoneal cells were collected by centrifugation. In patients with uremia who were undergoing CAPD, overnight (8-hour) PDE (glucose 1.36%) samples were collected and the volume was measured. The total volume of well-mixed PDE samples were centrifuged at 1600 rpm for 10 minutes at room temperature. Cell-free PDE samples were collected and stored at - 7 0 ° C until analysis for HA or cytokines. Blood samples were taken from these patients, at the time when the collection of PDE samples was completed, for determination of plasma and PDE concentrations of creatinine, albumin, IgG, and HA. The plasma level of HA was also determined in 26 healthy control subjects who were comparable in sex and age. Sandwich enzyme-linked immunosorbent assays were used to measure IL-l[3, IL-6, TGF-[31, or bFGF in the ascitic fluid or in PDE (R & D Systems, Minneapolis, Minn.). Detection limits of the assays are 0.03 pg/ml (IL-113), 0.06 pg/ml (IL6), 5 pg/ml (TGF-I]I), and 0.1 pg/ml (bFGF). HA in plasma or peritoneal fluid was measured by a radioimmunoassay with a detection limit of 10 gg/L (Pharmacia, Uppsala, Sweden). These cytokines and growth factors were measured in 20 of the 27 ascites samples. All samples were assayed at the same time to avoid inter-batch variation. Intra-assay coefficient of variation was less than 8% for all assays. The creatinine concentrations in ascitic fluid or PDE were measured by the alkaline-picrate method (Hitachi 911 analyzer; Boehringer Mannheim, Mannheim, Germany). The creatinine concentration in the PDE was calculated after correction of glucose interference with formulas determined by our previous studiesJ 3 Mesothelial cell and m a c r o p h a g e cultures• Mesothelial cells and macrophages were cultured to study (1) whether HA is synthesized by mesothelial cells and (2) whether macrophages can affect HA concentration in the ascitic fluid by either direct synthesis or by stimulating the mesothelial cells. Fractions rich in mesothelial cells or macrophages were prepared by countefflow centrifugal elution by using a JE-6B rotor (Beckman, Palo Alto, Calif.). In brief, 1 x 107 PDE cells in 5 ml of RPMI (Gibco, Chagrin Falls, N.Y.) were introduced into the elution rotor. The cells were loaded at a flow rate of 8.5 ml/min under room temperature with the rotor speed at 1500 rpm. After all cells were loaded, the rotor speed was set to 1000 rpm and varied by no more than 10 rpm during the procedure. The flow rate was then changed to 9 ml/min, and a 300 ml fraction was collected (F1). This was followed by a
356
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T a b l e I. The c o r r e l a t i o n c o e f f i c i e n t s (Rs) b e t w e e n p l a s m a a n d p e r i t o n e a l c o n c e n t r a t i o n s of c r e a t i n i n e ,
a l b u m i n , IgG, a n d HA Patients with cirrhosis* (n = 27)
Creatinine Albumin IgG HA
Patients undergoing CAPDt (n = 33)
0.97 (p = 0.0001) 0,46 (p = 0.02) 0.12 (p = 0,540) 0.09 (p = 0.657)
0.52 (p = 0.0033) 0.21 (p = 0.232) O.O7 (p = 0,694) 0.24 (p = 0.183)
Correlation coefficients determined by Spearman rank correlation. *Ascitic fluid. tPeritoneal dialysate effluent.
further increase to 85 ml/min, and a 200 ml fraction was collected (F2). The elution rotor was then stopped while the flow rate remained, and the remaining cells eluted and were designated the rotor-off fraction. Each fraction was washed in RPMI-1640. The composition of the cells in each fraction was analyzed by flow cytometry with fluorescein isothiocyanate-conjugated anti-human CD45 and RPE-conjugated anti-human CD14 or fluorescein isothiocyanate-conjugated anti-human cytokeratin (Dako, Kyoto, Japan). The F1 fraction contains more than 70% of macrophages and was designated the macrophage-rich fraction. The F2 fraction contains the highest proportion of mesothelial cells (>5%) and was named the mesothelial cell-rich fraction. Macrophages were isolated from the F1 fraction by adhesion. The cells were adjusted to 2 × 106 per ml RPMI-1640, were added to a 75 cm 2 flask, and were incubated for 1 hour at 37 ° C in a 5% CO 2 incubator. The nonadherent cells were discarded and the flask was washed twice to remove any residual nonadherent cells. Ninety-nine percent of the adhered cells were CD45- and CD14-positive as determined by flow cytometry. Mesothelial cells were isolated from the F2 fraction. Macrophages were removed by adhesion as described above. The nonadherent cells were collected and washed twice with RPMI-1640 and cultured overnight in complete culture medium (RPMI- 1640 supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine (2 retool/L), penicillin (100 U/ml), and streptomycin (100 gg/ml)(Gibco). The nonadherent cells were discarded, and 98% of the remaining adhered cells were cytokeratin-positive as determined by flow cytometry. Isolated mesothelial cells or macrophages were seeded at subconfluency level and were cultured for 1 week with 6-well plates. The mesothelial cells or macrophages were then subcultured at different cell concentrations for 36 hours with (1) culture medium or (2) macrophage-conditioned medium prepared by incubating separate batches of macrophages with complete culture medium for 72 hours. The cell supernatants were collected and kept at -70 ° C for assay of HA. Statistics. All results were expressed as median and range
unless specified otherwise. The data were analyzed with nonparametric tests, either by the Mann-Whitney test or the Spearman rank correlation, as appropriate. All probability values are two-tailed. RESULTS HA levels in plasma a n d in peritoneal fluid. The median
H A level in plasma from our 27 patients with cirrhosis (459 gg/L, range 154 to 1216) was significantly higher than that of CAPD patients (162 gg/L, range 30 to 1036, p < 0.0001) or healthy subjects (27.4 gg/L, range 22.4 to 39.1, p < 0.0001)(Fig. 1). Similarly, the H A level in ascites in our cirrhotic subjects was significantly higher than that o f PDE from patients with uremia who were undergoing C A P D (p = 0.0001). The patients with cirrhosis had a lower plasma level of albumin yet a higher albumin level in ascites than the plasma (23.8 gin/L, range 16.7 to 37.4 gin/L, versus 28.9 gin/L, range 19.9 to 38.3 gin/L, p -- 0.0296) o r P D E (3.79 gin/L, range 1.14 to 21.1 gin/L, versus 0.79 gm/L, range 0.02 to 15.1 gin/L, p = 0.0001) from patients with uremia who were undergoing CAPD. Both the plasma and peritoneal levels o f creatinine in patients with uremia who were undergoing CAPD (1173 gmol/L, range 533 to 1853 gmol/L, and 461 ~tmol/L, range 94 to 1700 ~tmol/L, respectively) were higher than values from patients with cirrhosis (110 gmol/L, range 58 to 1305 gmol/L, and 90 gmol/L, range 9 to 958 gmol/L, p = 0.0001 for both). There was a good correlation between the ascites and plasma levels of creatinine or albumin among our patients with cirrhosis but not in H A or IgG (Table I). In patients with uremia who were undergoing CAPD, similar correlation only existed between PDE and plasma levels of creatinine. Interleukin 1~, IL-6, TGF-131, and b F G F were measured in 20 of the 27 ascites samples. A significant correlation was observed between ascites level in H A and I L - t ~ , IL-6, or T G F - ~ I (Fig. 2) but not in b F G F (Rs =
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the 33 CAPD patients had a PDE/plasma ratio <1 (p = 0.0007, Fisher's exact test). These peritoneal/plasma ratios determined in patients with cirrhosis were always higher than those of patients with uremia who were undergoing CAPD. Mesothelial cell and macrophage cultures. Table III depicts the supematant concentrations of HA in mesothelial cells or macrophages cultured under different experimental conditions. HA is synthesized by both mesothelial cells and macrophages, although the former synthesizes twice the amount released by the latter. There was a stepwise increase in HA with increased cell number of ctfltured cells. There was an additive effect in the synthesis of HA by mesothelial cells when the macrophage-conditioned medium was added to the culture medium. DISCUSSION
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Fig. 2. A, B, and C depict a significant correlation between the HA levels and the IL-1 [3, IL-6, and TGF-~ concentrations in the ascitic fluid.
0.14, p = 0.583). There was no correlation between the ascitic levels of TGF-~I and bFGF (Rs = 0.28,p = 0.186). Peritoneal/plasma ratios of creatinine, albumin, IgG, and HA. The median peritoneal/plasma ratios of creatinine, albumin, and IgG in either cirrhotic or C A P D patients were less than unity (Table I). In contrast, the median peritoneal/plasma ratios of H A in both groups of patients exceeded one. Only one of the 27 patients with cirrhosis had an ascites/plasma ratio <1, yet 14 of
HA, which forms a critical component of extracellular matrixes, is present in high concentrations in tissues undergoing remodeling and morphogenesis, where it acts as a support for cell adhesion and locomotion. HA diffuses from ground substance and synovial membranes and enters the circulation via the lymphatics. It is rapidly cleared by a receptor-mediated process by hepatic endothelial cells. Renal excretion accounts for less than 1% of total body clearance of H A in normal subjects. 14 Serum HA is increased in inflammatory diseases such as rheumatoid arthritis, psoriatic arthropathy, and scleroderma and in certain malignancies.3, 4 Under these conditions it is likely that the increased levels reflect augmented synthesis by target cells such as fibroblasts, which are stimulated by various cytokines.4,15 In liver disease or chronic kidney failure, the increase in serum H A is thought to be the result of impaired turnover.5,12,16,17 H A is also found in PDE from patients undergoing CAPD.18,19 In this study we confirm the previous observation that patients with cirrhosis have raised plasma H A and that these values are higher than those in patients with uremia who were undergoing CAPD or those in comparable healthy controls. It was not surprising that the peritoneal/plasma ratios of creatinine, albumin, IgG, and H A in patients with cirrhosis were always higher than those in patients with uremia who were undergoing CAPD, because an equilibrium would not be reached after an 8-hour overnight dwell of dialysate. More interestingly, we have found that there is a higher concentration of H A in the ascitic fluid than in the plasma. The raised H A in the ascites from these patients was unlikely to derive from the systemic circulation because of diffusion across the peritoneal membrane. In such a case, one would also expect a correlation between the ascites and plasma levels of HA as demonstrated in creatinine and albumin. The failure to detect such a rela-
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Table II. The peritoneal/plasma ratios of creatinine, albumin, IgG, and HA Patients with cirrhosis* (n = 27) Creatinine
Patients undergoing CAPDt (n = 33)
0.83 (0.12-1.65) 0.15 (0.04-0.90) 0.10 (0:004-0.61) 2.54 (0.9-9.67)
Albumin IgG HA
p value
0.45 (0.10-1.13) 0.01 (0.005-0.61) 0.01 (0.006-0.39) 1.13 (0.41-13.71 )
0.0006 0.0001 0.0001 0.0035
All results are expressed as median and range, *Ascitic fluid, 1-Peritoneal dialysate effluent.
Table III. HA levels (gg/L) in mesothelial cell or m a c r o p h a g e cell culture Culture medium RPMI RPMI RPMI RPMI + M a c r o p h a g e conditioned medium
Cell number 1 1 1 1
x x x x
104 105 106 105
Mesothelial cells
Macrophages
83.4 201.8 511.8 333.5
19.8 92.5 210.7 101.0
+ + + +
7.42 19.97 34.64 28.48
_+ 4.13 _+ 17.84 + 25.19 + 14.22
Results are expressed as mean _+SEM for six experiments, The HA level in cell-free RPMI culture medium was <10 gg/L,
tionship and a high ascites/plasma ratio of H A - - i n contrast to findings with creatinine, albumin, and I g G - strongly suggest that there may also be intrinsic production of HA in these patients with cirrhosis by peritoneal cells or the hepatic stellate cells. Animal studies reveal increased biosynthesis of HA by the hepatic stellate cells after liver regeneration after acute liver failure or during endotoxemia. 20,21 Nevertheless, neither of these two conditions operated in our patients with cirrhosis, thus pointing to the possibility that the biosynthesis of HA was increased in the peritoneal cells. Our present study casts doubt on the previous hypothesis that the increase in serum HA among patients with chronic liver disease is a result of impaired turnover.SA 2 The renal clearance is trivial and is unlikely to contribute significantly, because all but two patients have normal renal function. Reduced degradation of HA cause by impaired function of liver endothelial cells leads to elevated plasma level of HA but not necessarily a raised ascitic concentration of HA. We have demonstrated by in vitro studies that HA is synthesized by human mesothelial cells isolated from ascites. In addition, we also found that macrophages synthesize HA with an amount that is half that needed by the mesothelial cells. There was an additive effect in the synthesis of HA by mesothelial cells when the macrophage-conditioned medium was added to the culture medium. These find-
ings are in accord with those of Yung et al., 19 who observed synthesis of HA by cultured peritoneal mesothelial cells isolated from patients undergoing CAPD. However, the biosynthesis of HA by peritoneal macrophages from CAPD subjects was not studied by these investigators. 19 Our present study strongly suggests that there is a simultaneous increased synthesis of HA by the peritoneal cells and a reduction in degradation by liver endothelial cells in patients with cirrhosis with ascites. The reason for an increased synthesis of HA by peritoneal cells leading to a raised ascitic level is most perplexing. Hamura et al. 22 have observed the accumulation of HA in an experimental model of peritonitis, and this event is in some way related to IL-1. An attractive mechanism for the augmented ascitic levels is an up-regulation of HA synthetase in the peritoneal mesothelial cells that are stimulated by proinflammatory cytokines (e.g., IL-1 [3, IL-6) released by the peritoneal macrophage after a subclinical or low-grade peritonitis. 23 IL-6 is produced in high amounts in the peritoneal cavity of patients with cirrhotic ascites, even in the absence of infection. 24 Either cytokine is known to stimulate HA production. 25 This hypothesis is supported by our observation that the level of HA correlates with either IL-1 [3 or IL-6 in the ascites. The function of HA in the peritoneal cavity and the consequences of high peritoneal levels remain uncertain. Being a critical component of extracellular
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matrixes with the ability to influence cell differentiation, proliferation, and aggregation, H A is l i k e l y to play an important pathophysiologic role in tissue repair in our patients with cirrhosis. The correlation between H A and T G F - ~ in ascites is most intriguing. T G F - ~ is bifunctional; that is, it can inhibit as well as stimulate cell growth. 26,27 Its effect on the proliferation of mese n c h y m a l cells is m o r e c o m p l i c a t e d . TGF-13 is both i n h i b i t o r y and s t i m u l a t o r y to fibroblast p r o l i f e r a t i o n under different p a t h o p h y s i o l o g i c environments. 28 A s t i m u l a t o r y effect of T G F - ~ on p e r i t o n e u m w o u l d favor fibroblast proliferation with increased deposition of e x t r a c e l l u l a r matrix, l e a d i n g to p e r i t o n e a l fibrosis and, subsequently, ultrafiltration failure in the peritoneal membrane. On the contrary, an inhibitory action o f TGF-[~ in ascites on p e r i t o n e a l fibroblasts m a y r e d u c e the risk of p e r i t o n e a l fibrosis/sclerosis and hence maintains a satisfactory peritoneal permeability. We speculate that the TGF-[3 in the ascites exerts an inhibitory action on the fibroblast, while H A p l a y s a contributory role in the r e m o d e l i n g and regeneration of the peritoneal lining. REFERENCES
1. Mason RM. Recent advances in the biochemistry of hyaluronan. Prog Clin Biol Res 1981;54:87-112. 2. Lanrent TC, Fraser JRE. Hyaluronan. FASEB J 1992;6:2397404. 3. Fosang AJ, Hey NJ, Carney SL, Hardingham TE. An ELISA plate assay for hyaluronan using biotinylated proteoglycan G1 domain. Matrix 1990;10:305-13. 4. Engstrom-Laurent A, Feltelius N, Hallgren R, Waterson R Elevated serum hyaluronate in scleroderma. An effect of growth factor induced activation of connective tissue cells. Ann Rheum Dis 1985;44:614-20. 5. Engstrom-Laurent A, Loof L, Nyberg A, Schroder T. Increased serum levels of hyaluronate in liver disease. Hepatology 1985;5:638-42. 6. Nyberg A, Engstrom-Laurent A, Loof L. Serum hyaluronate in primary biliary cirrhosis--a biochemical marker for progressive liver damage. Hepatology 1988;8:142-6. 7. Frebourg T, Delpech B, Bercoff E, Senant J, Bertrand P, Deugnier Y, et al. Serum hyaluronate in liver diseases: study by enzymo-immunological assay. Hepatology 1986;6:392-5. 8. Plebani M, Giacomini A, Floreani A, Chiaramonte M, Soffiati G, Naccarto R, et al. Biochemical markers of hepatic fibrosis in primary biliary cirrhosis. Res Clin Lab 1990;20:269-74. 9. Guechot J, Loria A, Serfaty L, Giral P, Giboudeau J, Poupon R. Serum hyaluronan as a marker of liver fibrosis in chronic viral hepatitis C: effect of a-interferon therapy. J Hepatology 1995;22:22-7. 10. Adams DH, Wang L, Hubscher SG, Neuberger JM. Hepatic endothelial cells, targets in liver allograft rejection? Trans-
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Hyaluronan (HA) is a polysaccharide that forms a critical component of extracellular matrixes. It is present in high concentrations in tissues undergoing remodeling and morphogenesis. Serum HA is elevated in patients with chronic liver disease, and this has been considered to be caused by impaired degradation by the liver endothelial cells. We studied the level of HA in the ascitic fluid and plasma from 27 patients with cirrhotic ascites. These values were compared with peritoneal dialysate effluent (PDE) and plasma from 33 patients with uremia who were undergoing continuous ambulatory peritoneal dialysis (CAPD). The median HA levels in ascitic fluid and plasma from our 26 patients with cirrhosis were significantly higher than corresponding PDE and plasma values from the 33 CAPD patients (p < 0.0001). The median peritoneal/plasma ratios of creatinine, albumin, and immunoglobulin G in either cirrhotic or CAPD patients were less than unity. In contrast, the median peritoneal/plasma ratios of HA in both groups of patients exceeded one with a higher peritoneal/plasma ratio of HA in patients with cirrhosis (p = 0.0035). A significant correlation was observed between the ascitic level of HA and interleukin-I~, interleukin-6, or transforming growth factor-~. Our in vitro cell culture studies revealed that HA is synthesized by both mesothelial cells and macrophages. We observed an additive effect in the synthesis of HA by mesothelial cells when the macrophage-conditioned medium was added to the RPMI culture medium. We conclude that a high level of HA is found in ascites from patients with cirrhosis. Our results strongly suggest that simultaneous increased synthesis of HA by the peritoneal cells and a reduction of degradation by liver endothelial cells occur in these patients with cirrhosis with ascites. This event of increased HA synthesis may be contributory to remodeling and regeneration of the peritoneal lining. (J Lab Clin Med 1998; 131:354-9) Abbreviations: bFGF = basic fibroblast growth factor; CAPE) = continuous ambulatory peritoneal dialysis; HA = hyaluronan; IgG = immunoglobulin G; IL = interleukin; PDE = peritoneal dialysate effluent; TGF-~= transforming growth factor-~
H
yaluronan is an unbranched high-molecularweight anionic polysaccharide that forms a critical component of extracellular matrixes. ~ HA can be synthesized by most cells and is present in From the Department of Medicine, Queen Mary Hospital, University of Hong Kong; and the Departments of Medicine and Chemical Pathology, Prince of Wales Hospital, Shatin. Supported by Grant HKU 4210/97M from the Research Grant Council and by the Thomas and Rita Liu Research Fund. Submitted for publication June 17, 1997;revision submitted Nov. 24, 1997; accepted Dec. 4, 1997. Reprint requests: K. N. Lai, Department of Medicine, University of Hong Kong, Room 409, Professorial Block, Queen Mary Hospital, Pokfulam, Hong Kong. Copyright © 1998 by Mosby, Inc. 0022-2143/98 $5.00 + 0 5/1/87994
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high concentrations in tissues undergoing remodeling and morphogenesis, where it acts as a support for cell adhesion and locomotion. It has also been shown to influence cell differentiation, proliferation, and aggregation, and it appears to play a critical role in the early stages of wound healing. 2-4 Some of the H A produced in the peripheral tissues is carried from the tissues by lymph flow to systemic circulation. Most of the HA is degraded in the lymph nodes, but a small percentage reaches the bloodstream, from where it is rapidly taken up by the liver endothelial cells and is degraded. 2 The serum concentration of HA is increased in chronic liver disease and has shown to be a predictor of development of cirrhosis. 5-9 The measurement of serum H A has also been shown to be useful in the monitoring of graft function after liver transplantation. 10,11 Rejection
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Fig. 1. Comparison of peritoneal concentration, plasma concentration, and peritoneal]plasma ratio of HA between 27 patients with cirrhosis with ascites (O) and 33 patients with uremia who were undergoing CAPD (O). These values are significantly higher in patients with cirrhosis with ascites.
episodes h a v e b e e n associated with a clear rise in H A . M e t a b o l i c studies w i t h injected H A have indicated that the liver m a y be a major site for the uptake of circulating H A in h u m a n beings. 12 A n elevated s e r u m H A level in patients with chronic liver diseases is considered to be the sequel of impaired clearance by liver endothelial cells.5,12 In this study w e h a v e e x a m i n e d the ascitic l e v e l o f H A in patients w i t h cirrhosis, and the source o f H A in ascites was explored. METHODS Patients• The study included 27 randomly selected patients with cirrhosis with ascites (20 men and 7 women, aged 57.8 + 14.9 years [mean __ SD]). Cirrhosis was diagnosed either by liver biopsy or clinical features of portal hypertension. The subjects were admitted for symptomatic treatment of ascites with no evidence of bacterial peritonitis. None of these patients had hepatic encephalopathy at the time of our study. The causes of chronic liver disease were chronic hepatitis B virus infection (19), chronic hepatitis C virus infection (6), and alcoholism (2). All but two of these patients with cirrhosis had normal renal function tests. None of these patients had paracentesis or bacterial peritonitis within 4 months of the study period. Thirty-three patients with uremia (20 men and 13 women, aged 55.6 +_ 11.3 years [mean + SD]) undergoing stable CAPD treatment and 26 healthy subjects (17 men and 9 women, aged 52.5 + 14.2 years [mean _+ SD]) served as control subjects. Patients with uremia in whom peritonitis developed within 4 months of the study period were excluded. Informed consent was obtained for collection of ascites and blood samples with approval of our hospital ethical committee. Determination of HA, IL-I [~, IL-6, TGF-[~, and bFGF concentrations in ascitic fluid or PDE. A 50 ml sample of ascitic fluid was collected under aseptic conditions from each cir-
355
rhotic patient on admission before any alteration of medication. The ascitic fluid was centrifuged at 1600 rpm for 10 minutes at room temperature. Cell-free ascitic fluid was collected and stored at -70 ° C until analysis for HA and cytokines. A blood sample was taken at the time of ascitic fluid collection for determination of the plasma and ascitic concentrations of creatinine, albumin, IgG, and HA. Ascitic fluid samples were examined to ensure absence of microbes or malignant cells. Therapeutic paracentesis with removal of 1 liter of ascitic fluid was also performed in 12 patients on admission, and peritoneal cells were collected by centrifugation. In patients with uremia who were undergoing CAPD, overnight (8-hour) PDE (glucose 1.36%) samples were collected and the volume was measured. The total volume of well-mixed PDE samples were centrifuged at 1600 rpm for 10 minutes at room temperature. Cell-free PDE samples were collected and stored at - 7 0 ° C until analysis for HA or cytokines. Blood samples were taken from these patients, at the time when the collection of PDE samples was completed, for determination of plasma and PDE concentrations of creatinine, albumin, IgG, and HA. The plasma level of HA was also determined in 26 healthy control subjects who were comparable in sex and age. Sandwich enzyme-linked immunosorbent assays were used to measure IL-l[3, IL-6, TGF-[31, or bFGF in the ascitic fluid or in PDE (R & D Systems, Minneapolis, Minn.). Detection limits of the assays are 0.03 pg/ml (IL-113), 0.06 pg/ml (IL6), 5 pg/ml (TGF-I]I), and 0.1 pg/ml (bFGF). HA in plasma or peritoneal fluid was measured by a radioimmunoassay with a detection limit of 10 gg/L (Pharmacia, Uppsala, Sweden). These cytokines and growth factors were measured in 20 of the 27 ascites samples. All samples were assayed at the same time to avoid inter-batch variation. Intra-assay coefficient of variation was less than 8% for all assays. The creatinine concentrations in ascitic fluid or PDE were measured by the alkaline-picrate method (Hitachi 911 analyzer; Boehringer Mannheim, Mannheim, Germany). The creatinine concentration in the PDE was calculated after correction of glucose interference with formulas determined by our previous studiesJ 3 Mesothelial cell and m a c r o p h a g e cultures• Mesothelial cells and macrophages were cultured to study (1) whether HA is synthesized by mesothelial cells and (2) whether macrophages can affect HA concentration in the ascitic fluid by either direct synthesis or by stimulating the mesothelial cells. Fractions rich in mesothelial cells or macrophages were prepared by countefflow centrifugal elution by using a JE-6B rotor (Beckman, Palo Alto, Calif.). In brief, 1 x 107 PDE cells in 5 ml of RPMI (Gibco, Chagrin Falls, N.Y.) were introduced into the elution rotor. The cells were loaded at a flow rate of 8.5 ml/min under room temperature with the rotor speed at 1500 rpm. After all cells were loaded, the rotor speed was set to 1000 rpm and varied by no more than 10 rpm during the procedure. The flow rate was then changed to 9 ml/min, and a 300 ml fraction was collected (F1). This was followed by a
356
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Lai et al.
T a b l e I. The c o r r e l a t i o n c o e f f i c i e n t s (Rs) b e t w e e n p l a s m a a n d p e r i t o n e a l c o n c e n t r a t i o n s of c r e a t i n i n e ,
a l b u m i n , IgG, a n d HA Patients with cirrhosis* (n = 27)
Creatinine Albumin IgG HA
Patients undergoing CAPDt (n = 33)
0.97 (p = 0.0001) 0,46 (p = 0.02) 0.12 (p = 0,540) 0.09 (p = 0.657)
0.52 (p = 0.0033) 0.21 (p = 0.232) O.O7 (p = 0,694) 0.24 (p = 0.183)
Correlation coefficients determined by Spearman rank correlation. *Ascitic fluid. tPeritoneal dialysate effluent.
further increase to 85 ml/min, and a 200 ml fraction was collected (F2). The elution rotor was then stopped while the flow rate remained, and the remaining cells eluted and were designated the rotor-off fraction. Each fraction was washed in RPMI-1640. The composition of the cells in each fraction was analyzed by flow cytometry with fluorescein isothiocyanate-conjugated anti-human CD45 and RPE-conjugated anti-human CD14 or fluorescein isothiocyanate-conjugated anti-human cytokeratin (Dako, Kyoto, Japan). The F1 fraction contains more than 70% of macrophages and was designated the macrophage-rich fraction. The F2 fraction contains the highest proportion of mesothelial cells (>5%) and was named the mesothelial cell-rich fraction. Macrophages were isolated from the F1 fraction by adhesion. The cells were adjusted to 2 × 106 per ml RPMI-1640, were added to a 75 cm 2 flask, and were incubated for 1 hour at 37 ° C in a 5% CO 2 incubator. The nonadherent cells were discarded and the flask was washed twice to remove any residual nonadherent cells. Ninety-nine percent of the adhered cells were CD45- and CD14-positive as determined by flow cytometry. Mesothelial cells were isolated from the F2 fraction. Macrophages were removed by adhesion as described above. The nonadherent cells were collected and washed twice with RPMI-1640 and cultured overnight in complete culture medium (RPMI- 1640 supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine (2 retool/L), penicillin (100 U/ml), and streptomycin (100 gg/ml)(Gibco). The nonadherent cells were discarded, and 98% of the remaining adhered cells were cytokeratin-positive as determined by flow cytometry. Isolated mesothelial cells or macrophages were seeded at subconfluency level and were cultured for 1 week with 6-well plates. The mesothelial cells or macrophages were then subcultured at different cell concentrations for 36 hours with (1) culture medium or (2) macrophage-conditioned medium prepared by incubating separate batches of macrophages with complete culture medium for 72 hours. The cell supernatants were collected and kept at -70 ° C for assay of HA. Statistics. All results were expressed as median and range
unless specified otherwise. The data were analyzed with nonparametric tests, either by the Mann-Whitney test or the Spearman rank correlation, as appropriate. All probability values are two-tailed. RESULTS HA levels in plasma a n d in peritoneal fluid. The median
H A level in plasma from our 27 patients with cirrhosis (459 gg/L, range 154 to 1216) was significantly higher than that of CAPD patients (162 gg/L, range 30 to 1036, p < 0.0001) or healthy subjects (27.4 gg/L, range 22.4 to 39.1, p < 0.0001)(Fig. 1). Similarly, the H A level in ascites in our cirrhotic subjects was significantly higher than that o f PDE from patients with uremia who were undergoing C A P D (p = 0.0001). The patients with cirrhosis had a lower plasma level of albumin yet a higher albumin level in ascites than the plasma (23.8 gin/L, range 16.7 to 37.4 gin/L, versus 28.9 gin/L, range 19.9 to 38.3 gin/L, p -- 0.0296) o r P D E (3.79 gin/L, range 1.14 to 21.1 gin/L, versus 0.79 gm/L, range 0.02 to 15.1 gin/L, p = 0.0001) from patients with uremia who were undergoing CAPD. Both the plasma and peritoneal levels o f creatinine in patients with uremia who were undergoing CAPD (1173 gmol/L, range 533 to 1853 gmol/L, and 461 ~tmol/L, range 94 to 1700 ~tmol/L, respectively) were higher than values from patients with cirrhosis (110 gmol/L, range 58 to 1305 gmol/L, and 90 gmol/L, range 9 to 958 gmol/L, p = 0.0001 for both). There was a good correlation between the ascites and plasma levels of creatinine or albumin among our patients with cirrhosis but not in H A or IgG (Table I). In patients with uremia who were undergoing CAPD, similar correlation only existed between PDE and plasma levels of creatinine. Interleukin 1~, IL-6, TGF-131, and b F G F were measured in 20 of the 27 ascites samples. A significant correlation was observed between ascites level in H A and I L - t ~ , IL-6, or T G F - ~ I (Fig. 2) but not in b F G F (Rs =
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the 33 CAPD patients had a PDE/plasma ratio <1 (p = 0.0007, Fisher's exact test). These peritoneal/plasma ratios determined in patients with cirrhosis were always higher than those of patients with uremia who were undergoing CAPD. Mesothelial cell and macrophage cultures. Table III depicts the supematant concentrations of HA in mesothelial cells or macrophages cultured under different experimental conditions. HA is synthesized by both mesothelial cells and macrophages, although the former synthesizes twice the amount released by the latter. There was a stepwise increase in HA with increased cell number of ctfltured cells. There was an additive effect in the synthesis of HA by mesothelial cells when the macrophage-conditioned medium was added to the culture medium. DISCUSSION
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800
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Fig. 2. A, B, and C depict a significant correlation between the HA levels and the IL-1 [3, IL-6, and TGF-~ concentrations in the ascitic fluid.
0.14, p = 0.583). There was no correlation between the ascitic levels of TGF-~I and bFGF (Rs = 0.28,p = 0.186). Peritoneal/plasma ratios of creatinine, albumin, IgG, and HA. The median peritoneal/plasma ratios of creatinine, albumin, and IgG in either cirrhotic or C A P D patients were less than unity (Table I). In contrast, the median peritoneal/plasma ratios of H A in both groups of patients exceeded one. Only one of the 27 patients with cirrhosis had an ascites/plasma ratio <1, yet 14 of
HA, which forms a critical component of extracellular matrixes, is present in high concentrations in tissues undergoing remodeling and morphogenesis, where it acts as a support for cell adhesion and locomotion. HA diffuses from ground substance and synovial membranes and enters the circulation via the lymphatics. It is rapidly cleared by a receptor-mediated process by hepatic endothelial cells. Renal excretion accounts for less than 1% of total body clearance of H A in normal subjects. 14 Serum HA is increased in inflammatory diseases such as rheumatoid arthritis, psoriatic arthropathy, and scleroderma and in certain malignancies.3, 4 Under these conditions it is likely that the increased levels reflect augmented synthesis by target cells such as fibroblasts, which are stimulated by various cytokines.4,15 In liver disease or chronic kidney failure, the increase in serum H A is thought to be the result of impaired turnover.5,12,16,17 H A is also found in PDE from patients undergoing CAPD.18,19 In this study we confirm the previous observation that patients with cirrhosis have raised plasma H A and that these values are higher than those in patients with uremia who were undergoing CAPD or those in comparable healthy controls. It was not surprising that the peritoneal/plasma ratios of creatinine, albumin, IgG, and H A in patients with cirrhosis were always higher than those in patients with uremia who were undergoing CAPD, because an equilibrium would not be reached after an 8-hour overnight dwell of dialysate. More interestingly, we have found that there is a higher concentration of H A in the ascitic fluid than in the plasma. The raised H A in the ascites from these patients was unlikely to derive from the systemic circulation because of diffusion across the peritoneal membrane. In such a case, one would also expect a correlation between the ascites and plasma levels of HA as demonstrated in creatinine and albumin. The failure to detect such a rela-
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Lai e t al.
Table II. The peritoneal/plasma ratios of creatinine, albumin, IgG, and HA Patients with cirrhosis* (n = 27) Creatinine
Patients undergoing CAPDt (n = 33)
0.83 (0.12-1.65) 0.15 (0.04-0.90) 0.10 (0:004-0.61) 2.54 (0.9-9.67)
Albumin IgG HA
p value
0.45 (0.10-1.13) 0.01 (0.005-0.61) 0.01 (0.006-0.39) 1.13 (0.41-13.71 )
0.0006 0.0001 0.0001 0.0035
All results are expressed as median and range, *Ascitic fluid, 1-Peritoneal dialysate effluent.
Table III. HA levels (gg/L) in mesothelial cell or m a c r o p h a g e cell culture Culture medium RPMI RPMI RPMI RPMI + M a c r o p h a g e conditioned medium
Cell number 1 1 1 1
x x x x
104 105 106 105
Mesothelial cells
Macrophages
83.4 201.8 511.8 333.5
19.8 92.5 210.7 101.0
+ + + +
7.42 19.97 34.64 28.48
_+ 4.13 _+ 17.84 + 25.19 + 14.22
Results are expressed as mean _+SEM for six experiments, The HA level in cell-free RPMI culture medium was <10 gg/L,
tionship and a high ascites/plasma ratio of H A - - i n contrast to findings with creatinine, albumin, and I g G - strongly suggest that there may also be intrinsic production of HA in these patients with cirrhosis by peritoneal cells or the hepatic stellate cells. Animal studies reveal increased biosynthesis of HA by the hepatic stellate cells after liver regeneration after acute liver failure or during endotoxemia. 20,21 Nevertheless, neither of these two conditions operated in our patients with cirrhosis, thus pointing to the possibility that the biosynthesis of HA was increased in the peritoneal cells. Our present study casts doubt on the previous hypothesis that the increase in serum HA among patients with chronic liver disease is a result of impaired turnover.SA 2 The renal clearance is trivial and is unlikely to contribute significantly, because all but two patients have normal renal function. Reduced degradation of HA cause by impaired function of liver endothelial cells leads to elevated plasma level of HA but not necessarily a raised ascitic concentration of HA. We have demonstrated by in vitro studies that HA is synthesized by human mesothelial cells isolated from ascites. In addition, we also found that macrophages synthesize HA with an amount that is half that needed by the mesothelial cells. There was an additive effect in the synthesis of HA by mesothelial cells when the macrophage-conditioned medium was added to the culture medium. These find-
ings are in accord with those of Yung et al., 19 who observed synthesis of HA by cultured peritoneal mesothelial cells isolated from patients undergoing CAPD. However, the biosynthesis of HA by peritoneal macrophages from CAPD subjects was not studied by these investigators. 19 Our present study strongly suggests that there is a simultaneous increased synthesis of HA by the peritoneal cells and a reduction in degradation by liver endothelial cells in patients with cirrhosis with ascites. The reason for an increased synthesis of HA by peritoneal cells leading to a raised ascitic level is most perplexing. Hamura et al. 22 have observed the accumulation of HA in an experimental model of peritonitis, and this event is in some way related to IL-1. An attractive mechanism for the augmented ascitic levels is an up-regulation of HA synthetase in the peritoneal mesothelial cells that are stimulated by proinflammatory cytokines (e.g., IL-1 [3, IL-6) released by the peritoneal macrophage after a subclinical or low-grade peritonitis. 23 IL-6 is produced in high amounts in the peritoneal cavity of patients with cirrhotic ascites, even in the absence of infection. 24 Either cytokine is known to stimulate HA production. 25 This hypothesis is supported by our observation that the level of HA correlates with either IL-1 [3 or IL-6 in the ascites. The function of HA in the peritoneal cavity and the consequences of high peritoneal levels remain uncertain. Being a critical component of extracellular
J Lab Clin Med Volume 131, Number 4
matrixes with the ability to influence cell differentiation, proliferation, and aggregation, H A is l i k e l y to play an important pathophysiologic role in tissue repair in our patients with cirrhosis. The correlation between H A and T G F - ~ in ascites is most intriguing. T G F - ~ is bifunctional; that is, it can inhibit as well as stimulate cell growth. 26,27 Its effect on the proliferation of mese n c h y m a l cells is m o r e c o m p l i c a t e d . TGF-13 is both i n h i b i t o r y and s t i m u l a t o r y to fibroblast p r o l i f e r a t i o n under different p a t h o p h y s i o l o g i c environments. 28 A s t i m u l a t o r y effect of T G F - ~ on p e r i t o n e u m w o u l d favor fibroblast proliferation with increased deposition of e x t r a c e l l u l a r matrix, l e a d i n g to p e r i t o n e a l fibrosis and, subsequently, ultrafiltration failure in the peritoneal membrane. On the contrary, an inhibitory action o f TGF-[~ in ascites on p e r i t o n e a l fibroblasts m a y r e d u c e the risk of p e r i t o n e a l fibrosis/sclerosis and hence maintains a satisfactory peritoneal permeability. We speculate that the TGF-[3 in the ascites exerts an inhibitory action on the fibroblast, while H A p l a y s a contributory role in the r e m o d e l i n g and regeneration of the peritoneal lining. REFERENCES
1. Mason RM. Recent advances in the biochemistry of hyaluronan. Prog Clin Biol Res 1981;54:87-112. 2. Lanrent TC, Fraser JRE. Hyaluronan. FASEB J 1992;6:2397404. 3. Fosang AJ, Hey NJ, Carney SL, Hardingham TE. An ELISA plate assay for hyaluronan using biotinylated proteoglycan G1 domain. Matrix 1990;10:305-13. 4. Engstrom-Laurent A, Feltelius N, Hallgren R, Waterson R Elevated serum hyaluronate in scleroderma. An effect of growth factor induced activation of connective tissue cells. Ann Rheum Dis 1985;44:614-20. 5. Engstrom-Laurent A, Loof L, Nyberg A, Schroder T. Increased serum levels of hyaluronate in liver disease. Hepatology 1985;5:638-42. 6. Nyberg A, Engstrom-Laurent A, Loof L. Serum hyaluronate in primary biliary cirrhosis--a biochemical marker for progressive liver damage. Hepatology 1988;8:142-6. 7. Frebourg T, Delpech B, Bercoff E, Senant J, Bertrand P, Deugnier Y, et al. Serum hyaluronate in liver diseases: study by enzymo-immunological assay. Hepatology 1986;6:392-5. 8. Plebani M, Giacomini A, Floreani A, Chiaramonte M, Soffiati G, Naccarto R, et al. Biochemical markers of hepatic fibrosis in primary biliary cirrhosis. Res Clin Lab 1990;20:269-74. 9. Guechot J, Loria A, Serfaty L, Giral P, Giboudeau J, Poupon R. Serum hyaluronan as a marker of liver fibrosis in chronic viral hepatitis C: effect of a-interferon therapy. J Hepatology 1995;22:22-7. 10. Adams DH, Wang L, Hubscher SG, Neuberger JM. Hepatic endothelial cells, targets in liver allograft rejection? Trans-
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