lmmunopharmacology~ 24 (1992) 181-190 © 1992 Elsevier Science Publishers B.V. All rights reserved 0162-3109/92/$05.00

181

IMPHAR 00603

Tamm-Horsfall glycoprotein (THG) purified from normal human pregnancy urine increases phagocytosis, complement receptor expressions and arachidonic acid metabolism of polymorphonuclear neutrophils Chia-Li Yu a, Whu-Mei Lin a, Tang-Shueen Liao ~, Chang-Youh Tsai b, Kuang-Hui Sun c and K u e n - H o r n g Chert d a Section of Allergy, Immunology and Rheumatology, Department of Medicine, Veterans General Hospital. bInstitute of Clinical Medicine. c School of Medical Technology, National Yang-ll~_'.':~g" Medical College and d Division of Rheumatologv, Cathay General Hospital, Taipei, Taiwan, ROC

(Received 10 February 1992; accepted 7 May 1992)

Abstract: Tamm-Horsfall glycoprotein (THG) purified from normal h~man pregnancy urine was found to increase polymorphonuclear neutrophil (PMN) phagocytosis (46.57 _+3.54% in the medium versus 75.85 _+5.37% in the presence of 25 #g/ml THG) after 30 min preincubation. The phagocytosis-enhancing activity of THG was dose-dependent (5-50 #g/ml) and was possibly mediated by the increased expressions of complement receptor type 1 (CRI) and type 3 (CR3) on the neutrophils. The release of [SH]arachidonic acid and prostaglandin E 2 (PGE2), but not thromboxane B2 (TXB2), from neutroph~!s were also significantly enhanced by THG. Using 3,3'-dihexyloxacarbocyanine iodide as indicator, THG (25 pg/ml) depolarized the membrane potential of PMN after 30 min preincubation. In addition, THG exhibited a specific membranotropic effect with PMN. It is conceivable that THG binds to the cell surface and depolarizes the membrane potential of PMN which subsequently enhances the release of arachidonic acid metabolites and the translocation of the complement receptors to the membrane. These biochemical events lead to the increment of PMN phagocytosis and suggests that THG may play an important role in the defense mechanisms of the urinary tract in that a large amount of THG is usually present.

Key words: Tamm-Horsfall glycoprotein; Phagocytosis; Complement receptor; Arachidonic acid; Membrane potential

Introduction Correspondence to: Chia-Li Yu, Section of Allergy, Immunology and Rheumatology, Department of Medicine, Veterans General Hospital-Taipei, National Yang-Ming Medical College, # 20! Section 2, Shih-Pai Road, Taipei, Taiwan I 1217, ROC. Abbreviations: THG, Tamm-Horsfalt glycoprotein; PMN, Polymorphonuclear neutrophil; CR1, complement receptor type 1; CR3, complement receptor type 3; AA, arachidenic acid; PGE 2, prostaglandin E2; TXB2, thromboxane B2.

Tamm-Horsfall glycoprotein (THG), a 7 x i07 kDa macromoiecule, is synthesized exclusively by renal tubular cells (Cornelius et al., 1965; Hoyer et al., 1974) and is normally found in the urine (Downay et al., 1982; Hunt et al., 1985), amniotic fluid (Rose et al., 1975; Phimister and Marshall, 1982) and serum (Avis, 1977; Hunt et al., 1986). The relative abundance, spe-

182 cific nephronal location and evolutional conservation of THG suggest that this glycoprotein possesses an important physiologic function as a 2CI--K +-Na + co-transporter in the rei, al interstitium (Budi-Santoso et al., 1987). Immunologically, Hunt and McGiven (1978) found that THG per se could stimulate human peripheral lympbocyte proliferation as if it wzre a nonspecific mitogen. However, in the presence of antigens or mitogens (Muchmore, 1986; Muchmore et al., 1987)o THG markedly suppressed lymphocyte proliferation. Recently, Kuriyama and Silverblatt (1986) found that THG decreased serum-independent phagocytosis and bactericidal activity of PMN via its anti-opsonic effect. Nevertheless, Horton and coworkers (1990) demonstrated that THG activated inflammatory responses of PMN and led to marked interstitial tissue damage in the case of tubular obstruction. The diverse effects of THG on immunological and inflammatory reactions seem dependent on the different functional domains of the macromolecule (Williams et al., 1984; Dall'Olis et al., 1988), but the real mechanism has still not been explored. In the present study, THG was purified from normal human pregnancy urine and the biochemical events involved in the priming of PMN by THG were investigated.

NaC1, centrifuged, and the precipitate washed twice with 0.58 M NaC1. This precipitate was dissolved in alkaline distilled water (pH 9.0), stirred overnight at 4 ° C, again centrifuged, and any precipitate discarded. The solution was dialysed extensively against distilled water at 4 °C, lyophilized and suspended in RPMI-1640. The purity and relative molecular weight (mw) of the obtained glycoprotein was analysed by 12.5~o S D S - P A G E under reducing conditions. The electrophoretic voltage was set at 100 mV and the temperature was set at 4 °C. The total electrophoresis time was 2 h.

Isolation of PMN from normal human peripheral blood Heparinized venous blood obtained from normal individuals was mixed with one-quarter of a volume of 2 ~ dextran solution (mw 500 000) and incubated .'t 37°C for 20m in. Leukocyteenriched supernatant was collected and diluted with the same volume of Hanks' balanced salt solution (HBSS). The cell suspension was gently layered over FicolI-Hypaque cushion (specific gravity 1.077) and centrifuged at 300 x g for 30 rain. The PMN were collected from the bottom. The contaminated RBC were lysed with cold 0.85~ NH4C1 solution. The purity of PMN was ~eater than 95~o as confirmed by Wright's stain.

Materials and Methods

Purification of THG from normal human pregnancy urine A twenty-four-h urine was collected form normal pregnancy after the sixth month. The separation of THG from urine was carried out by the method reported by Hunt and McGiven (!978) with modifications. Briefly, batch urine was made up to 0.58 M with NaCI and stirred for 30 min, centrifuged at 2000 × g for I0 min and the supernatant discarded. The precipitate was dissolved in alkaline distilled water (pH 9.0 was adjusted by the addition of i N NaOH) and again centrifuged to remove any insoluble material. The supernatant was then made up to 0.58 M with

Measurement of PMN phagocytosis by flow cytometry Fluoresbrit carboxylate microspheres (0.75 #m; Polysciences Inc., Warrington, PA, USA) were previousiy opsonized with fresh normal human serum at 37 °C for 45 rain. PMN "3 x 106/ml) were preincubated with different concentrations of THG (5-.50 itg/ml) for 30 rain before mixing with opsonized beads (1 x 108/ml). The percentage and fluorescence intensity of PMN with phagocytosis were measured by EPICS C flow cytometry (Coulter Electronics Inc., Hialeah, FL, USA) with 488 nm excitation as in our previous report (Yu et al., 1988). Because THG at a concentration of 25/~g/ml exhibited a maximal

183 PMN phagocytosis enbancing effect, this concentration was used in most of the experiments.

Measurements of complement receptor type 1 (CR1) and type 3 (CR3) expressions of PMN Indirect immunofluorescence antibody method was used to stain the CR1 and CR3 on PMN. Monoclonal mouse anti-human CD35 (CR1) and CD1 lb (CR3) (Dako Incorp., Dakopatts, Denmark) were the primary antibodies. FITC-labeled rabbit anti-mouse IgG2a antibodies were the secondary antibodies° P M N (1 x 106/ml) were preincubated with T H G (25 #g/ml) at 37 °C for 30 rain belbre staining. The receptors on PMN was measured by EPICS C flow cytometry with 488 nm excitation. To verify that the upregulated CR1 and CR3 on PMN by T H G were specific, mouse IgG2a (Sigma Chemical Co., St. Louis, MO, USA) was used as primary antibody and the following staining procedures were the same as the method described above.

the suspension in an ice-bath. The cells were then centrifuged at 400 x g at 4 °C for 10 rain. Half the volume of the supematant was transferred into a counting vial filled with 3 ml aqueous scintillation fluid, and radioactivity was measured by Packard TriCarb 2000 CA liquid scintillation counter. Results were presented as cpm/2× 106 PMN. The total count was the sum of cpm in the supernatant and pellet in the control tube.

Detection of the binding capacity of THG with PMN Half a milliliter of PMN suspension (2 x 106/ml) was mixed with 0.5 ml RPMI-1640 or T H G (50/ag/ml) and incubated at 37°C in 5% CO2-95~/0 air for 60 rain. After washing three times with medium, the cells were stained with FITC conjugated sheep anti-THG antibodies (FITC-anti-uromucoid; The Binding Site Lt, University of Birmingham Research Institute, Birmingham, UK). The percentage and fluores-

THG -1

Estimation of membrane potentials of &dividual cells We followed the method described by Shapiro et al. (1979). The indicator dye used was 3,3'dihexyloxacarbocyanine iodide (Eastman Kodak, Rochester, NY). The membra~qe potential of neutrophils was measured after preincubation with T H G (25/ag/ml) for 30 min by EPICS C flow cytometry with 488 nm excitation.

THG -2

67-.!¸

Measurement of [SH]arachidonic acid releasefi'om PMN We followed the method reported by Atkinson et al. (1990) with modifications. Briefly, 0.5 ml PMN (1 × 107/ml) was incubated with 20/zl [3H]arachidonic acid (100/tCi/ml; NEN Products, Boston, MA, USA) at 37 °C for 60 rain. After washes in cold 0.1 )~, bovine serum albumin (BSA) in RPMI medium, 0.25 ml aliquots of PMN (2 × 106/ml) were then individually incubated with 0.25 ml 0.1% BSA in RPMI (control tube), T H G (25/ag/ml),, A23187 ( 2 # M ) or FMLP (1 x 10 -7 M) at 37 °C for 45 rain. After incubation, the reaction was stopped by placing

30-Fig. 1. ElectrophoreticanalysisofTamm-Horsfallglycoprotein in 12.5% SDS-PAGE under reducing conditions. A major band with a relativemolecularweightof 94-100 kDa was found.

184 TABLE I Effects of T H G (25 ttg/ml) on the percentage of phagocytosis, complement receptor type i (CR 1) and type 3 (CR3) expressions, and membrane potential of PMN cells after preincabation for 30 rain P M N cells (~o) n=8

Phagocytosis

CR 1

CR3

Membrane potential

P M N + medium

46.57 + 3.54 (67.29 _+3.76 # )~ 75.85 _+5.37 (81.06 + 5.81 # ) 0.0013 b

94.56 + 2.08 (90.43 _+4.93 # ) 95.07 + 1.89 (137.89 + 7.67# ) 0.0034 ¢

90.16 +_ 1.87 (114.07 + 3.86 # ) 94.17 _+ 1.78 (150.71 _+3.20# ) 0.041 c

100 + 0 (74.21 + 5.43 # ) 100 + 0 (58.66 + 4 . 1 7 # ) 0.0042 ¢

PMN + T H G P

~ The value in parentheses represents mean fluorescence intensity measured by EPICS C flow cytometry. b p value was calculated by comparing the percentage of phagocytosis in P M N + medium and P M N + T H G by non-paired Student's t-test. c p va!ue was calculated by comparing the mea~ fluorescence intensity of P M N + medium and PMN + T H G by non-paired Student's t-test.

PHAGOCYTO$1S

PHAGOCYTOSIS 2, PMN~'Agg.~-GLOBULIN

1, PMN~HBSS

( IOOuglmD

.J

L

L

ffl ,,,,I

w U tL 0

3

~

~

'

.. , r

4,PMN~.THG(25ug/ml)

PMN ÷THG ( l O u g / m D

W

M X Z

, ~

/

m

l

,

~ ,, FLUORESCENCEINTENS;TY

)

FLUORESCENCEINTENSITY Fig. 2. Dose-dependent effect of Tamm-Horsfall glycoprotein (THG) on the neutrophil phagocytosis after preincubation for 30 min. (1)Phagocytosis of P M N preincubated with HBSS = 60.58~'o; mean fluorescence intensity (MFI)= 59.78 # . (2)Phagocytosis of P M N prehlcubated with human heat aggregated ~,-globulin (100 ~Lg/ml)= 60.97%; MFI = 63.01 # . (3) Phagocytosis of PMN preineubated with T H G (10#g/ml)=68.24~o; M F I = 5 8 . 8 6 # . (4)Phagocytosis of P M N preincubated with T H G (25 #g/ml) = 83.36%; ,~dFl = 66.09#. (5) Phagocytosis of P M N preincubated with T H G (50 t~g/ml) = 77.36~o; MFI = 61.96 # .

185 cence intensity of the positively stained cells were detected by EPICS C flow cytometry (Coulter Electronics). In order to confirm the binding specificity of T H G with PMN, T H G was previously incubated with 1:20 diluted antiuromucoid antiserum (The Binding Site Lt, Birmingham, UK) or anti-dsDNA antibodies (370 IU/ml) purified from active SLE sera in our laboratory at 37°C for 60min before adding to the PMN suspension.

ELISA for prostaglandin E2 (PGE2) and thromboxane B2 (TXB2)

30 rain. The cell-free supernatant was obtained after centrifugation at 3 0 0 x g for 10 min. The concentration of P G E 2 and TXB2 in these supernatants was measured by commercially available EIA kits (Cayman Chemical Co. Inc, Ann Arbor, MI, USA). The low detection limit was 3.9 pg/ml in PGE 2 and 7.8 pg/ml in TXB2 respectively.

Statistical analysis Results represent the mean + S.D. in this study. Statistical significance was assessed by Student's t-test (paired or non-paired).

One milliliter of PMN (2 x 106/ml in PBS, pH 7.2) reacted with T H G (25 #g/mi) at 37 °C for

CR3

CR1

in .,J .J t/J

t~

k

1.pMN+2°Ab

4 . PMN+ ;~°Ab

a . PMN+HBSS

5 . PMN+HBSS

3 . PMN+THG

6.PMN+THG

l

u.

0 ¢1: UJ

en E Z

FLUORESCENCE

INTENSITY

Fig. 3. Effects ofTamm-HorsfaUglycoprotein (THG)on the expressions of complement receptor type 1 (CRl,left panel) and type 3 (CR3, right panel) on the neutrophils after preincubating with HBSS or THG (25/~g/ml) for 30 mkl. (1) PMN was stained with secondary antibodies only = 1.52%. (2)CR1 expression after preincubating with HBSS = 97.37~; mean fluorescence intensity (MFI)= 104.66#. (3) CR1 expression after preincubating with T H G = 9 7 . 3 7 ~ ; M F I = t18.33#. (4) PMN was stained with secondary antibodies only = 2.46%. (5) CR3 expression after preincubating with HBSS = 93.61~; MFI = 143.98 # . (6) CR3 expression after preincubating with THG = 97.83 %; MF! = 157.69 # .

186

MEMBRANE POTENTIAL

Results

I. PMN~HBSS

THG puri~d ftvm normal human pregnancy urine A m z v ~ m o l e c u l e was obtained from urine sampled during prebq~ancy aider repeated precipitation/dissolution in 0.58 M NaC1/alkaline distilled water. The relative molecular weight o f T H G was estimated at 94-100 k D a by 12.5Yo S D S - P A G E analysis, as shown in Fig. 1. Functionally, the macromolecule was capable o f stimulating lymphocyte proliferation at concentrations of > 10 #g/ml with a maximal effect at 25 #g/ml in 3-day culture (data not shown). Accordingly, 2 5 # g / m l o f T H G was used in most 9f the experiments.

°t

~ "---~

z~.PMN.FMLP

|

.=~

Effect of THG on the phagocytosis and complement receptor expressions on PMN Preincubation of T H G (25 #g/ml) with P M N for 30 mia significantly increased the phagocytosis o f P M N (46.57+3.54~o in the medium versus 75.85+5.37~o in th~ presence o f T F I G ; P = 0 . 0 0 1 3 ) (Table I). A typical case with a dose-dependent effect o f T H G (10, 25 and 50 #g/ml) is presented in Fig. 2. Because T H G exhibits a low solubility (about 0.5 ~o) in solution with a neutral p H and tends to be gel-like, we only used the soluble form of T H G after centrifuging and filtrating the T H G suspension through a 0.22-#m millipore filter. In addition, heataggregated (63 °C for 45 miru '-uman ~,-globulin

FLUORESCENCEINTENSITY Fig. 4. Membrane potential change of neutrophil after stimulation with Tamm-Horsfall glycnprotein(THG 25 #g/ml) or FMLP (1 × 10 -7 M) for 30 min. (1) Positive cell = 99.54%, mean fluorescence intensity (MFI)= 85.06#. (2) Positive cell = 99.72%; MFI = 61.73 #. (3) Positive cell = 98.44~; MFI=61.81#.

( 1 0 0 # g / m l ) was used as control in case the residual particulate T H G or aggregated T H G formed in the salt precipitatioii method i~ the T H G supernatant might non-specifically stimu-

TABLE II The binding capacity of THG (25 #g/ml) with PMN detected by flow cytometry Exp no.

1 2 3 4 5

% Binding (mean fluorescence intensity) PMN + RPMI

PMN + THG

PMN + THG + anti-uromucoid"~

PMN + THG + anti-dsDNA b

5.31 (44.83#) 5.47 (92.86# ) 4.43 (70.39#) 5.15 (76.83#) 2.65(61.47 # )

89.33 (104.51 #) 92.49 (137.86#) 93.66 (151.52#) 86.47 (121.45#) 94.74(116.65# )

8.63 (56.86# ) 12.03 (87.43 #) 10.56 (72.34#) 7.42 (73.59# ) 11.89(81.17# )

74.86 (102.31 #) 88.56 (140.94#) 93.67 (143.86#) 90.41 (122.35#) 86.36(108.71 # )

a THG was preincubated with 1:20 diluted sheep anti-human uromucoid antiserum at 37 °C for 60 min before reacting with PMN. THG was preincubated with 370 IU/ml of anti-dsDNA antibodies purified from pooled SLE sera at 37 °C for 60 min before reacting with PMN.

187 late phagocytosis (Fig. 2). To explore the mechanism of increased phagocytosis by TIIG, the expressions of CR1 and CR3 on PMN were measured using flow cytometry. The fluorescence intensity (receptor density) of both CR1 and CR3 on PMN was significantly increased by THG (Table I). A typical case is demonstrated in Fig. 3.

Effect of THG on the membrane potential change of PMN THG (25/~g/ml) depolarized the membrane potential of PMN after pretreatment for 30 rain (fluorescence intensity = 74.21 _+5.43 # in medium-treated PMN versus 58.66 + 4.17 # in THG-treated PMN, P=0.0042) (Table I). A typical case is shown in Fig. 4. The binding capacity of THG with PMN THG exhibited a strong binding affinity with the surface membrane of PMN, as shown in Table II. This binding capacity could be abrogated by the specific anti-uromuc6id aittibody, but not by human anti-dsDNA antibodies (370 IU/ml). The experiments were repeated five times and a similar tendency was observed. Effects of THG on the release of arachidonic acid, PGE: and TXBe from PMN THG is able to activate phospholipase and subsequently hydrolyse cel[ membrane phospholipid, causing arachidonic acid release (Table III).

TABLE Ill Arachidonic acid (AA) release from PMN after incubation with medium, THG (25 !tg/m!), FMLP (1 x !0 ~ 7 M) and A23187 (2 ILM) for 30 min Exp no.

1 2 3 4

Release of AA (cpm)

Total count (cpm)

Medium

THG

FMLP

A23187

1473 1058 1320 916

4242 5856 4000 3146

5209 5524 4248 3554

16703 19240 20484 9520

28843 32898 51998 20430

,L The total count is the sum of cpm in the supernatant and pellet in the control tube in which 0.25 ml of 0.1% bovine serum albumin in RPM1 was added instead of 0.25 ml of other active reagents.

Four experiments were repeated to confirm the results. In addition, the generation of PGE 2, but not TXBz, was also increased by THG stimulation (Table IV).

Discussion

Tamm-Horsfall glycoprotein, a major constituent of urinary cast, is the most abundant protein in normal human urine. In the present study, we purified a factor with a molecular weight of 94-100 kDa from normal human urine samples during pregnancy. Functionally, this molecule could directly stimulate lymphocyte proliferation

TABLE IV Generation of prostaglandin E 2 (PGE2) and thromboxane B 2 (TXI32) by neutrophils after incubation with HBSS, THG (25/tg/ml) and A23187 (2 #M) for 30 min Concentration in the culture supernatant (pg/ml) n = 10

PGE 2

P

TXB 2

P

Medium THG A23187

882.87 _+330.44 1990.00 + 436.57 8374.00 +_960.63

0.00046 a 0.00000 b

224.15 +_125.27 317.63 + 314.74 1097.50 + 520.52

0.454 a 0.00145 ~

a Compare T H G with medium; P value was calculated by paired Student's t-test. b Compare A23187 with medium; P value was calculated by paired Student's t-test.

188 but suppressed the proliferation of mitogen or antigen stimulated lymphocyte (data not shown). These properties are quite similar to Tamm-Horsfall glycoprotein described by Itunt and McGiven (1978) or uromodulin reported by Muchmore et al. (1986, 1987) although we did not further purify the factor through Con A-Sepharose and Fractogel 55 S columns and measured the cytokin inhibitory activities of tbe molecule. Some authors have demonstrated that THG could directly bind with viruses (Tamm etal., 1952, 1955) and bacterial extracts (Schachner et al., 198'7) and thereby abrogate the invasiveness of pathogenic microbes in the urinary tract. Recently, an increasing amount of evidence indicates that THG is the renal ligand for cytokines (Hession etal., 1987) including IL-1, IL-2 and TNF-~ (Brown etal., 1986; Muchmore, 1986; Muchmore and Decker, 1986; Sherblom et al., 1988, 1989; Winkelstein et al., 1990). In addition to these immunologic activities, Horton etal. (1990) demonstrated that THG can activate the inflammatory responses of neutrophils such as respiratory burst, degranulation, enzymes secretion, and leukotriene B 4 generation. These findings indicate that THG potentiates the immunologic and inflammatory reactions particularly in the urinary system. In the present study, three interesting biological effects of THG on PMN were found: (a)THG bound to the surface membrane of PMN and depolarized the membrane potential of these cells; (b) THG enhanced PMN phagocytosis and translocated the complement receptors from the cytoplasm to the surface membrane of PMN; and (c)THG activated the arachidonic acid metabolism of the PMN. Using FITClabeled anfi-uromucoid antibodies as probe, THG was demo~strated to be able to bind with PMN, and this binding could be: blocked by the previous treatment of THG with anti-THG antibodies but not with unrelated antibody such as human anti-dsDNA (Table II). This membranebinding activity of THG not only depolarized the membrane potential of cells but enhanced at least two biological activities of PMN: phagocytosis

and the release of arachidonic acid metabolites. The increased phagocytosis of PMN was accompanied by upregulated CR1 and CR3 expressions. Because we did not measure the expression of Fc receptor on PMN after incubating with THG, the real role of complement and Fc receptors in mediating the enhancement of PMN phagocytosis by THG is not clear. However, both receptors are believed to be essential for the binding and ingestion of particles by PMN. Our result seems different from that of Kuriyama and Silverblatt (1986) in that phagocytosis and bacterial killing of PMN were suppressed by THG when type-1 fimbriated E. coli were the targets. Although the real cause for the difference between our results and those of Kuriyama and Silverblatt is not clear, the different assay methods used in these two studies probably account for it. They used mannose-sev~itive (type 1) fimbriated E. coli as targets which were previously coated with THG. This coating formed a pseudocapsule on E. coli and allowed bacteria escaping from serum-independent phagocytosis to be killed by host PMN in the urinary tract. However, we measured PMN phagocytosis by preincubating PMN with THG for 30 rain and then reacted it with opsonized beads. The preincubation of PMN with THG not only translocated the complement receptors from cytoplasm to surface membrane but activated phospholipase. The enzyme hydrolyses the membrane phospholipid and elicits the release of arachidonic acid and PGE2 but not TXB 2 from PMN. The released PGE 2 further recruits the inflammatory and immunologic reactive cells to the site of lesion to enforce the inflammatory reaction. In order to further confirm this, the determination of phospholii.~ase A2 and the enzymes involved in the cyclooxygenase and lipooxygenase pathways in THG-acti~,ated PMN is now under investigation. Pathologically, interstitial deposits of THG have been reported in obstructive uropathy (Dziukas etal., 1982), renal transplant rejection (Howie and Brewer, 1983; Cohen et al., 1984), nephrolithiasis (Resniek et al., 1978; Zager et at.,

189 1978) and in the glomerular mesangium in experimental models of vesico-ureteric reflux in rats. These reports suggest a possible involvement of THG in glomertdar scarring associated with reflux nephropathy (Mackenzie and Asscher, 1986). Our results further confirm that THG may serve as an amplified loop of inflammatory reaction in the urinary tract in that levels of complements and inflammatory cytokins are extremely low, but a high concentration of THG is usually present. In conclusion, the membranotropic activity of THG depolarizes the membrane potential of PMN causing release of arachidonic acid metabolite~;, translocation of complement receptors and iacreased phagocytosis. These events contribute to the defense mechanism of the urinary tract. Acknowledgements This work was supported by grants from the National Science Council (NSC 81-0412-B07512), ROC, the Institute of Biomedical Sciences, Academia Sinica, and the Ching-Ling Resem'ch Foundation for Geriatric Medicine (CI-80-15). We are grateful to Miss Tzu-Miao Lin and Miss Yi-Sheng Chert for their technical assistance. We are also indebted to Miss Chun-Shia Chong for typing the manuscript. References Atkinson YH, Murray AW, Krilis S, Vadas MA, Lopez AF. Human tumour necrosis factor-alpha (TNF-e) directly stimulates arachidonic acid release in human neutrophils. Immunology 1990; 70: 82-87. Avis PJG. The development of radioimmunoassay procedure for the estimation of Tamm-Horsfall glycoprotein in human serum. Clin Sci Mol Med 1997; 52: 183-t91. Brown KM, Muchmore AV, Rosenstreich D L Uromodulin, an immunosuppressive protein derived from pregnancy urine, is an inhibitor of interleukin-l. Proc Natl Acad Sci USA t986; 83: 9119-9123. Budi-Santoso AW, Scott DM, Kinne R. Localization of Tamm-Horsfall protein in chloride transporting epithelia: lack of correlation with Na +, K +, C1- cotransporter. Eur J Ceil Biol t987; 43: 104-109.

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