Polarized expression of Tamm-Horsfall protein by renal tubular epithelial cells activates human granulocytes

Abstract

In renal bacterial infections granulocytes are of major importance in the primary immune defense against invading pathogens. However, the mechanisms of granulocytic activation in renal interstitial invasion have not been clarified. Renal tubular epithelial cell mechanisms inducing granulocytic activation and bacterial killing may include tubular cell expression of Tamm-Horsfall protein (THP), a urinary protein that is known to enhance cytokine expression in monocytes. We studied the role of THP in granulocytic activation. A strong binding of THP to human granulocytes was demonstrated by fluorescence-activated cell sorter analysis. Urinary THP and supernatants of THP-expressing cultured tubular epithelial cells (MDCK) enhanced interleukin-8 (IL-8) expression by human granulocytes. Renal tubular cells growing polarized on polycarbonate membranes were used to study apical versus basal THP expression. By electron microscopy THP immunoreactivity was exclusively found on the apical surfaces of tubular cells and was absent on the basolateral cell membrane. In the apical cell culture compartment we found significantly more stimulatory activity for granulocytic IL-8 expression. CD62L, a selectin less expressed in activated granulocytes, was decreased in granulocytes incubated with urinary THP and in supernatants of THP-producing renal tubular cells but not in supernatants from THP-negative cells. Again, the effect on CD62L expression was found only in apical culture media and was absent in the basal compartment. In summary our data give evidence that renal tubular cell THP expression may be relevant in kidney diseases since THP is a potent activator of human granulocytes. The regulation of apical versus basal THP expression and release in vivo may be crucial in the induction of the inflammatory response, e.g., in bacterial renal diseases.

FIG. 1.

Immunohistochemical detection of THP expression in a human kidney section. Normal human renal tissue was snap-frozen and stained for THP expression by immunohistochemistry as described in Materials and Methods. No staining was observed in glomeruli and vessels, but a fraction of tubular epithelial cells strongly stained for THP. Original magnification, ×100.

FIG. 2.

Postembedding immunogold labeling for THP in MDCK:THP⁺ (A) and MDCK:THP⁻ (B) cells. MDCK cells were grown on polycarbonate membranes and were embedded in LR white. Note the prevailing label on the apical membrane and microvilli in panel A. Bars: 150 nm.

FIG. 3.

FACS analysis of THP binding to granulocytes. THP binding to human granulocytes was detected by FACS analysis as described in Materials and Methods. Granulocytes were incubated with THP (1 ng/ml, 100 ng/ml, and 10 μg/ml) or in the presence of MDCK:THP⁺ or MDCK:THP⁻ cell culture supernatants. Dotted-line histograms, controls for THP binding without addition of THP-containing samples; solid-line histograms, THP binding, as described in Materials and Methods. Out of three independent experiments one histogram is shown per condition.

FIG. 4.

(A) Urinary THP stimulates IL-8 expression in granulocytes. Shown is the effect of THP dose (10 ng/ml to 10 μg/ml) on IL-8 expression by human granulocytes. Granulocytes were incubated with various concentrations of THP as indicated or LPS (1 μg/ml) for 24 h, and IL-8 expression was detected by ELISA (n = 3 determinations/bar). In the presence of anti-THP (right bar) the effect of THP (1 μg/ml) was reduced to control levels. (B) Stimulation of IL-8 by apical versus basal supernatants of MDCK:THP⁺ and MDCK:THP⁻ cells. MDCK:THP⁺ and MDCK:THP⁻ cells were cultured on polycarbonate microtiter plates, and the apical and basal cell culture media were harvested separately. IL-8 expression by human granulocytes was measured by ELISA as described in Materials and Methods. Control granulocytes were incubated with cell culture medium only. Bars show means of six determinations/column plus standard deviations. ∗, P < 0.05 versus value for THP⁺ basal medium; ∗∗, P < 0.01 versus value for controls or MDCK:THP⁻ apical cell supernatants (U test).

FIG. 5.

(A) Urinary THP reduces granulocytic CD62L expression. CD62L expression in human granulocytes was detected as described in Materials and Methods. In the presence of LPS (1 μg/ml) and in the presence of urinary THP (1 μg/ml) CD62L expression was reduced. Dotted-line histograms, conjugate controls; open histograms, CD62L expression; solid histograms, CD62L expression after sample incubation, as described in Materials and Methods. Out of three independent experiments one histogram is shown per condition. (B) Apical and basal culture media of MDCK:THP⁺ and MDCK:THP⁻ cells differentially reduce CD62L expression in granulocytes. Apical and basal cell culture media were collected separately, and the effect on granulocytic CD62L expression was analyzed. Dotted-line histograms, conjugate controls; open histograms, CD62L expression; solid histograms, CD62L expression after sample incubation, as described in Materials and Methods. Out of three independent experiments one histogram is shown per condition.