Effects of Tamm–Horsfall protein on polymorphonuclear leukocyte function

Thomas Wimmer, Gerald Cohen, Marcus D. Saemann and Walter H. Hörl

Division of Nephrology and Dialysis, Department of Medicine III, University of Vienna, Vienna, Austria

Correspondence and offprint requests to: Walter H. Hörl MD, PhD, FRCP, Division of Nephrology and Dialysis, Department of Medicine III, Währinger Gürtel 18–20, A-1090 Vienna, Austria. Email: walter.hoerl{at}nephro.imed3.akh-wien.ac.at



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Tamm–Horsfall protein (THP), a glycoprotein produced exclusively by renal tubular cells, is thought to be involved in several inflammatory disorders such as bacterial interstitial nephritis as well as in defence against uropathogenic microorganisms. The specific effects of THP on inflammatory cells, however, are not yet well known. Therefore, the present study investigates the effects of THP in its soluble form on distinct polymorphonuclear leukocyte (PMNL) functions.

Methods. PMNL were isolated from the venous blood of healthy adult donors and incubated at low THP concentrations (70–350 ng/ml), resembling plasma concentrations, and at high THP concentrations (1.75–8.75 µg/ml), resembling urinary concentrations.

Results. High (urinary) THP concentrations inhibited PMNL apoptosis and chemotaxis and stimulated PMNL phagocytosis, while low (plasma) THP concentrations increased PMNL chemotaxis.

Conclusions. These data indicate that THP influences several PMNL functions, suggesting a crucial immunomodulatory role for this glycoprotein in host defence mechanisms of the kidney and genitourinary tract.

Keywords: apoptosis; chemotaxis; phagocytosis; polymorphonuclear leukocytes; Tamm–Horsfall protein



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Tamm–Horsfall protein (THP) or uromodulin is synthesized exclusively by the thick ascending limb of the loop of Henle and in the distal convoluted tubule of the kidney [1]. The main part of the molecule is a polypeptide containing 639 amino acids, and ~30% of the molecular weight is carbohydrates [2]. THP belongs to the family of glycosylphosphatidylinositol (GPI)-anchored proteins [3] and is expressed on both the luminal and basolateral poles of the cell membrane [4]. As it is the most abundant protein in human urine, up to 200 mg are excreted daily under physiological conditions. Moreover, significant serum levels in healthy individuals have been described, ranging from 70 to 540 ng/ml [5,6].

The exact physiological role of THP, however, still remains enigmatic. Since THP is the matrix of all renal stones, it has been implicated in renal stone and cast formation [7–9]. THP may also play a role in defence against urinary tract infections, as it interferes with adherence of Escherichia coli to uroepithelial cells [10]. There is also evidence that THP may play a proinflammatory role within the kidney in several tubulointerstitial diseases [11]. Interstitial deposits of THP have been found surrounded by leukocyte infiltration [12,13]. Interestingly, recent data have shown that mutations in the THP gene are involved in hyperuricaemia and chronic interstitial renal disease [14–16].

The activation of lymphocytes by THP has been detailed in previous studies [17,18]. Regarding polymorphonuclear leukocytes (PMNL), Toma et al. [19] have characterized THP as a leukocyte adhesion molecule, while Horton et al. [20] described the interaction of particulate THP and PMNL. However, the detailed effects of THP on PMNL, which are involved in the early innate immune response, are still largely unknown. Therefore, the present study examines the effects of THP in its soluble form on particular PMNL functions using THP concentrations found locally in the kidney and urinary tract, as well as concentrations resembling physiological and pathological serum THP levels. We found that THP is involved in the modulation of chemotaxis, phagocytosis and spontaneous apoptosis, indicating an immunomodulatory role for this particular glycoprotein.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Tamm–Horsfall protein
THP isolated from normal human urine, purified by extensive precipitation and buffered in an aqueous solution with 0.02% sodium azide at a concentration of 350 µg/ml was purchased from Biotrend (Köln, Germany). Purity had been assessed through a single homogenous band on 6–7.5% SDS–PAGE at 92 kDa.

PMNL isolation
PMNL were isolated from venous blood of healthy adult donors of both genders (age range 20–45 years) using discontinuous Ficoll–Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. Venous whole blood was anticoagulated by collecting it into sterile 10 ml lithium heparin Vacutainer tubes (Vacuette®; Greiner bio-one, Kremsmünster, Austria), diluted 1:1 with 10 ml of 0.9% NaCl and then put on top of 12 ml of Ficoll–Hypaque (100%, density 1.077 g/ml, 20°C). After centrifugation, the whole supernatant, including the plasma layer, an interface of slow sedimenting particles (lymphocytes, monocytes and platelets) and the Ficoll–Hypaque layer, was decanted from a pellet consisting of PMNL and erythrocytes. Erythrocytes were removed from the pellet by hypotonic lysis with ammonium chloride buffer (157 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA Na2) on ice. After lysis, the PMNL were collected by centrifugation at 4°C and washed once with ammonium chloride buffer and twice with Dulbecco's phosphate-buffered saline (PBS; pH 7.2; Gibco-BRL Life Technologies). The final preparation was stored in PBS at 4°C until required (not longer than 1 h). The purity of the PMNL population obtained by this method was >90% (containing <10% other leukocytes, mainly lymphocytes) and verified by counting the cells in a cell counter (Cell Dyn 610; Abbott, Wiesbaden, Germany).

PMNL function tests
Chemotaxis
PMNL chemotaxis was determined by the under-agarose method [21]. N-formyl-methionyl-leucyl-phenylalanine (fMLP; Sigma, St Louis, MO) dissolved in Hank's buffer (at a concentration of 4 x 10–7 M) was used as a chemoattractant. PMNL were resuspended in PBS at a concentration of 5 x 105 cells/10 µl. During the PMNL migration step, the agarose plates were incubated for 2 h at 37°C. Then the cells were fixed with methanol and paraformaldehyde and stained with Giemsa (Merck, Darmstadt, Germany). The distance migrated under agarose was measured under the microscope.

To test the effect of THP, four different dilutions in PBS were included in the PMNL suspension and pre-incubated for 15 min at 37°C. The final concentrations of THP were adjusted to 70 and 350 ng/ml, and 1.75 and 8.75 µg/ml. For each concentration, 10–11 separate assays were performed.

Spontaneous apoptosis of PMNL
PMNL were isolated under sterile conditions as described above. The cell suspension (2 x 106 cells/ml in PBS) was incubated at 37°C for 20 h. As in the chemotaxis assay, four different final THP concentrations were adjusted at 70 and 350 ng/ml, and 1.75 and 8.75 µg/ml. Afterwards (i) morphological features; (ii) caspase-3 activity; and (iii) DNA content of the PMNL were analysed in order to assess PMNL spontaneous apoptosis.

  1. Morphological features: the PMNL suspension was mixed with fluorescent DNA-binding dyes and examined under the fluorescence microscope. Acridine orange (Merck, Darmstadt, Germany) and ethidium bromide (Gibco-BRL Life Technologies) were added to a final concentration of 5 µg/ml each. Acridine orange is taken up by the cells, binds to DNA by intercalation between stacked base pairs and appears green. Ethidium bromide is taken up only by dead cells without an intact cell membrane, also binds to DNA, but stains it orange to a stronger extent than acridine orange. As the DNA in non-apoptotic cells is structured within the nucleus and the DNA in apoptotic cells is condensed, the following four cell populations can be observed and counted under the microscope: (i) viable, non-apoptotic with a green, structured nucleus; (ii) apoptotic with a green, condensed nucleus; (iii) late apoptotic with an orange, condensed nucleus; and (iv) necrotic with an orange structured nucleus (usually not seen in this experimental setup). For each concentration, 9–15 separate assays were performed.
  2. Caspase-3 activity: activation of ICE-family proteases (caspases) initiates apoptosis in mammalian cells. Caspase-3, which plays a central role as effector caspase, is an intracellular cysteine protease that exists as a proenzyme, becoming activated during the cascade of events associated with apoptosis. Caspase-3 cleaves a variety of cellular molecules that contain the amino acid motif DEVD, such as poly(ADP-ribose)polymerase (PARP), a subunit of the DNA-dependent protein kinase. The presence of caspase-3 in cells of different lineages suggests that caspase-3 is a key enzyme required for the execution of apoptosis [22]. A colorimetric protease assay kit (Caspase-3 Colorimetric Assay; R&D Systems) based on the spectrophotometric detection (at a wavelength of 405 nm) of the chromophore p-nitroanilide (pNA) after cleavage from the labelled substrate DEVD-pNA was used to detect apoptosis. For each concentration, nine separate assays were performed.
  3. Analysis of DNA content: apoptotic cells have a lower DNA content as a result of DNA cleavage by an activated nuclease. The DNA content was analysed by flow cytometry. After 20 h incubation at 37°C, the PMNL (1.2 x 106/200 µl) were centrifuged at 360 g for 20 min and washed twice with PBS. A 250 µl aliquot of ice-cold 70% ethanol was added to the pellet. After 60 min incubation on ice, the PMNL were centrifuged, washed once with PBS and resuspended in 200 µl of PBS containing 250 µg/ml RNase (type I-A; Sigma) and 50 µg/ml propidium iodide (Sigma). After 15 min incubation at room temperature in the dark, samples were kept on ice in the dark until flow cytometric analysis. For each concentration, six separate assays were performed.

Control experiments: in order to show that the inhibiting effect upon PMNL apoptosis was not only an unspecific protein effect due to an added supply of substrate (considering that THP might serve as such) but a specific effect of the substance itself, we performed additional control experiments. To provide a sufficient supply of substrates, we incubated PMNL and THP (at 70 and 350 ng/ml, and 1.75 and 8.75 µg/ml) in RPMI 1640 medium (Gibco-BRL Life Technologies) containing 10% deactivated BCS (bovine calf serum, triple 0.1 µm sterile filtered, heat-deactivated at 42°C for 30 min; HyClone, Logan, UT) for 20 h. PMNL apoptosis was assessed by analysation of the DNA content after staining with propidium iodide as described above. For each concentration, eight separate assays were performed.

Phagocytosis
The quantitative determination of PMNL phagocytosis was performed in heparinized whole blood. Flow cytometry was used to determine the percentage of PMNL that had ingested fluorescein isothiocyanate (FITC)-labelled opsonized E. coli (Phagotest, Orpegen Pharma, Heidelberg, Germany) and the amount of ingested E. coli per PMNL in the absence and presence of THP at four different final concentrations, as used in the other neutrophil functional assays (70 and 350 ng/ml, and 1.75 and 8.75 µg/ml). For each concentration, 9–12 separate assays were performed.

Control experiments: in order to exclude a possible contamination of THP by bacterial lipopolysaccharide (LPS), THP was pre-incubated with polymyxin B (PMB; Sigma) at a concentration of 10 µg/ml at 37°C for 60 min. As a control, LPS (Sigma) at a concentration of 100 U/ml was pre-incubated with PMB under the same conditions.

Afterwards, PMNL phagocytosis was assessed as described above with samples containing (i) PBS only; (2) 8.75 µg/ml THP; (ii) 8.75 µg/ml THP pre-incubated with PMB 10 µg/ml; (iv) 10 µg/ml PMB only; (v) 100 U/ml LPS; and (vi) 100 U/ml LPS pre-incubated with PMB 10 µg/ml. Nine separate assays were performed.

Statistics
In the figures, the means and standard errors are given. Statistical analysis of all experiments was done using Wilcoxon's two-sided test for paired samples. Differences were considered significant, if P-values were <0.05.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Chemotaxis
The under-agarose method [21] was used to test the influence of four different THP concentrations on the chemotactic response of PMNL. We found that THP added to PMNL at a final concentration of 350 ng/ml, which resembles serum THP levels in healthy subjects [5], significantly increases the migration of PMNL towards fMLP (Figure 1). Using a final THP concentration of 70 ng/ml, which is similar to serum THP levels found in patients with impaired renal function [5], a significant increase in directed PMNL migration was observed (Figure 1). Yet there is no significant difference between either THP concentration.



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Fig. 1. Effects of THP at the chemotactic movement of PMNL from healthy subjects towards FMLP. N = 10. Mean value ±SEM. *P<0.01, **P<0.025, ***P<0.005.

 
Whereas serum THP levels increase PMNL movement towards fMLP, a significant inhibition of directed PMNL migration is seen at higher THP levels resembling urinary concentrations (1.75 and 8.75 µg/ml; Figure 1). THP itself when used as a chemoattractant instead of fMLP did not show any chemotactic effect (data not shown). Furthermore, THP, when added to fMLP did not affect the chemotactic activity of fMLP, excluding the possibility that the inhibiting effect of THP on PMNL chemotaxis is caused by direct binding of THP to fMLP (data not shown).

Spontaneous apoptosis of PMNL
To test the capability of THP to influence PMNL apoptosis, (i) morphological features; (ii) caspase-3 activity; and (ii) DNA content were assessed as described above.

First PMNL were stained with fluorescent DNA-binding dyes and examined under the fluorescence microscope. Three different PMNL populations were distinguished and counted by their morphological features: (i) viable, non-apoptotic cells; (ii) apoptotic cells; and (iii) late apoptotic cells. At final THP concentrations of 70 and 350 ng/ml, we found a significant decrease in the percentage of viable, non-apoptotic PMNL (Figure 2). The number of apoptotic cells increased. On the other hand, at higher THP concentrations (1.75 and 8.75 µg/ml), the opposite effect was observed: increased numbers of viable, non-apoptotic cells (Figure 2). The numbers of cells exhibiting typical features of apoptosis decreased.



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Fig. 2. Effects of THP on spontaneous apoptosis of PMNL from healthy subjects. Percentage of viable, non apoptotic cells by morphological features after 20 h incubation time. N = 18. Mean value ±SEM. *P<0.05, **P<0.005.

 
Furthermore, the activity of caspase-3 in PMNL, which is the central effector caspase triggering apoptotic cell death, was assessed through a colorimetric protease test kit and quantitated spectrophotometrically. Final THP concentrations of 1.75 and 8.75 µg/ml resulted in a significant decrease in caspase-3 enzymatic activity (Figure 3), indicating a decrease in PMNL apoptotic cell death. At low THP concentrations (70 and 350 ng/ml), no significant effect on PMNL caspase-3 activity was observed.



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Fig. 3. Effects of THP on spontaneous apoptosis of PMNL from healthy subjects. Caspase-3 activity measured after 20 h incubation. N = 9. Mean value ±SEM. *P<0.05.

 
Additionally, the DNA content of the PMNL was analysed by flow cytometry after staining with propidium iodide. At all THP concentrations tested (70 and 350 ng/ml, and 1.75 and 8.75 µg/ml), a decrease in apoptotic cells was observed, suggesting an inhibitory effect of THP on PMNL apoptotic cell death (Figure 4A).



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Fig. 4. Effects of THP on spontaneous apoptosis of PMNL from healthy subects. Analysis of DNA content after 20 h incubation in (A) PBS and (B) RPMI 1640 medium +10% FCS. N = 8 (A) N = 9 (B). Mean value ±SEM. *P<0.05.

 
To exclude an unspecific protein effect of THP, which might serve as a substrate for PMNL and thus prolong the life span and decrease the rate of apoptosis, we incubated the cells and THP in RPMI 1640 medium containing 10% heat-deactivated BCS for 20 h. Afterwards we analysed the DNA content of the PMNL as described above. We detected a significant decrease in PMNL apoptosis in samples containing THP vs the control group containing culture medium only; thus the effect of THP on PMNL apoptosis is substance specific and not an unspecific protein effect (Figure 4B).

Phagocytosis
PMNL phagocytosis was determined by flow cytometry after ingestion of FITC-labelled E. coli. Whereas THP concentrations of 70 and 350 ng/ml did not show a significant change in the percentage of PMNL that had ingested FITC-labelled bacteria (data not shown), we detected a significant increase in phagocytosing PMNL as well as in E. coli per PMNL using higher THP concentrations (1.75 and 8.75 µg/ml, Figure 5A).



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Fig. 5. Effects of THP on the phagocytic functions of PMNL obtained from healthy subjects. (A) Solid black bars showing the percent difference of E. coli per PMNL, grey bars the percent difference of PMNL phagocytosing compared to the control group. N = 12 (1.75 µg/ml). N = 18 (8.75 µg/ml). Mean value ±SEM. *P<0.05, **P<0.005, ***P<0.001. (B) Control experiments (percent difference of E. coli per PMNL compared to the control group shown) were done with Tamm-Horsfall protein 8.75 µg/ml (THP), THP 8.75 µg/ml preincubated with polymyxin B 10 µg/ml (THP + PMB), polymyxin B 10 µg/ml (PMB), lipopolysaccharide 100 U/ml (LPS) and LPS 100 U/ml preincubated with PMB 10 µg/ml (LPS + PMB). N = 9. Mean value ±SEM. *P<0.05, **P<0.01, n.s. not significant.

 
Since THP shows a similar effect on PMNL phagocytosis to that of LPS, we performed additional control experiments adding PMB, which prevents LPS binding to its receptor [23], to exclude LPS-induced effects on PMNL functions. While the stimulating effect of 100 U/ml LPS on PMNL phagocytosis (i.e. the amount of E. coli per PMNL) was completely neutralized by the addition of 10 µg/ml PMB (Figure 5B), there was no significant difference between the stimulation of PMNL phagocytosis (E. coli per PMNL) by 8.75 µg/ml THP and by 8.75 µg/ml THP pre-incubated with 10 µg/ml PMB (Figure 5B), indicating that the effect is mediated by THP itself and not by contamination with LPS. PMB itself did not have any significant effect on the amount of E.coli ingested (Figure 5B).



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The results presented in this study show that THP in its soluble form at concentrations similar to serum levels found in healthy subjects and in patients with impaired renal function [5], and at higher concentrations, which occur locally in the kidney and urinary tract [6], has the potential to modulate essential functions of PMNL. Higher levels of THP resembling urinary concentrations were observed to inhibit PMNL apoptosis and chemotaxis and to stimulate PMNL phagocytosis. Inhibition of apoptotic cell death and the increase in phagocytosis suggest an activating effect of THP on PMNL at these concentrations. The stimulating THP effect observed on PMNL phagocytosis at higher concentrations appeared to be similar to a response caused by bacterial LPS. Since THP is extracted from human urine, contamination with LPS would be possible. However, as determined by the respective experiments in the present study, significant contamination of the THP was ruled out, since the effect of THP on PMNL phagocytosis was not offset by polymyxin B, a typical and well-known deactivator of bacterial LPS.

On the other hand, THP at lower concentrations equalling physiological and pathological serum levels appeared to increase PMNL chemotaxis, suggesting an activating effect as well. However, the effects on apoptosis at low THP concentrations were controversial and PMNL showed a decrease in apoptotic DNA cleavage while the morphological characteristics displayed an increase in early apoptotic features. This might be explained by the fact that morphological features such as cell shrinking and chromatin condensation might be visible at an earlier stage of apoptosis before significant DNA cleavage occurs. Finally, PMNL phagocytosis was not influenced by lower THP concentrations.

The dose-dependent effect of THP on PMNL chemotaxis, acting in either a stimulatory (at low concentrations) or an inhibitory manner (at high concentrations), may play a role in the microenvironmental regulation of inflammatory processes. Low concentrations may enhance neutrophil migration towards the focus of inflammation, while higher doses cause the cells to remain at the site of inflammation. Several studies, recently summarized by Calabrese [24], illustrate the common biphasic nature of a broad range of chemoattractant and chemotaxis-enhancing molecules, with low doses usually enhancing and high doses inhibiting chemotaxis, independent of both the target tissue and chemoattractant agent. In the common underlying mechanism, most receptor systems use concentration gradients to implement the enhancement and inhibition of the regulatory message system. Therefore, agonists may activate different receptor subtypes at either low (nanomolar) or higher (micromolar) concentrations due to differences in receptor affinities, and act in either a stimulatory or an inhibitory manner. Also, the maximum stimulatory THP effect on various neutrophil functions, such as chemotaxis and phagocytosis, might be reached at different concentrations, thus showing no correlation at a particular dose.

The modulation of these particular PMNL functional characteristics by THP has not been described to date, but fits well with the results of previous studies. THP is a leukocyte adhesion molecule [19] that binds to a single class of carbohydrate-specific receptors on human neutrophils [4]. It initiates the inflammatory response of PMNL, including the activation of the respiratory burst as well as comprehensive PMNL degranulation by the particulate form of this protein [20]. Furthermore, THP increases complement receptor expression and arachidonic acid metabolism of PMNL [25]. Recently, Kreft et al. observed that THP expression by renal tubular epithelial cells activates PMNL, resulting in interleukin-8 (IL-8) expression [26].

Our results are in line with previous studies indicating that THP may be relevant for PMNL recruitment and activation in bacterial kidney and urinary tract infections. In this context, THP could influence neutrophil migration towards an inflammatory focus as well as phagocytosis of bacteria. Recently, it was shown that THP is able to bind to type 1 fimbriated E. coli [10], which could lead to opsonization and increased bacterial ingestion by PMNL. Therefore, we are tempted to speculate that THP plays a role in host defence in the genitourinary tract both through binding bacteria and therefore preventing them from adhering to epithelial cells, and through direct activation of PMNL.

THP may have a pathogenic role in the aetiology of several tubulointerstitial diseases especially during the stage of early infiltration by neutrophils. The capability to influence PMNL chemotaxis might be a factor in the attraction of neutrophils by interstitial THP deposits [11,13]. The modulation of PMNL spontaneous apoptosis by THP could have an effect on the replacement of the early infiltrate by mononuclear cells and lymphocytes during the progression of the response. THP might also play a systemic immunological role. Other studies entailed the effect of THP on tumour necrosis factor-{alpha} (TNF-{alpha}) production by human monocytes [18] and the promotion of cytokine clearance (IL-1 and TNF) by THP [27].

Our findings imply that normal serum levels of THP are sufficient to influence PMNL chemotaxis and spontaneous apoptosis. Yet there is no significant difference in PMNL response between serum THP levels found in healthy subjects and those found in patients with impaired renal function. Therefore, it is rather unlikely that the low serum THP levels in patients with renal diseases contribute to a decrease in immune defence. Nevertheless, a physiological role for THP in the systemic immune response regarding PMNL functions and life span might be considered.

In conclusion, our results indicate that THP influences several PMNL functions in a differential mode, suggesting an essential immunomodulatory role for THP in local host defence mechanisms of the kidney and genitourinary tract.

Conflict of interest statement. None declared.



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
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Received for publication: 8. 8.03
Accepted in revised form: 11. 2.04





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