Interleukin-1P and tumor necrosis factor-a synergistically stimulate nerve growth factor synthesis in rat mesangial cells PHILIPP STEINER, JOSEF PFEILSCHIFTER, CLEMENS BOECKH, HEINFRIED RADEKE, AND UWE OTTEN Department of Physiology, University of Base& and Research Department, Pharmaceuticals Division, Ciba-Geigy, CH-4051 Base& Switzerland; Department of Pharmacology, University of Freiburg, D-7800 Freiburg; and Department of Molecular Pharmacology, Hannover Medical School, D-3000 Hannover, Federal Republic of Germany

STEINER, PHILIPP, BOECKH, HEINFRIED

JOSEF PFEILSCHIFTER, RADEKE, AND UWE OTTEN.

CLEMENS

Interleukinstimulate nerve

lp and tumor necrosis factor-a synergistically growth factor synthesis in rat mesangial cells. Am. J. Physiol. 261 (Renal Fluid Electrolyte Physiol. 30): F792-F798, 1991.-

Recent evidence indicates that cytokines are potent inducers of nerve growth factor (NGF) expression both in peripheral tissues and the central nervous system and that NGF, in addition to its neurotrophic action, also acts asan immunoregulatory agent. It was of interest to investigate whether inflammatory cytokines affect NGF production in renal mesangial cells, which play a crucial role in the modulation of the local immune function in the glomerulus.Our results show that the simultaneous addition of interleukin-l@ (IL-lp) and tumor necrosisfactor-a) (TNF-(u) elicited a marked (13-fold) increase of NGF protein releasedby cultured rat glomerular mesangial cells within 24 h, whereasIL-la in combination with TNF-cu, as well as the cytokines alone, did not promote the synthesis of NGF. The synergistic effect was dosedependent (maximal at 1 nM) and due to enhanced gene expression, since the cytokine treatment causeda fivefold increase in NGF mRNA after 8 h. Stimulation of NGF synthesis was abolished by mepacrineand dexamethasone,indicating that phospholipase AZ may be involved in NGF regulation. Moreover, pretreatment of the cells with the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) abolishedinduction of NGF by cytokines; in contrast, the specific cyclooxygenaseinhibitors indomethacin and diclofenac failed to modify NGF production. These data suggestthat a lipoxygenasemetabolite produced in responseto IL-lp and TNF-ac acts as a mediator in NGF gene expression. In conclusion, these findings support a model in which a cytokine cascadeincluding NGF may play an important role in the pathophysiology of inflammatory renal diseases. glomerular mesangium;cytokines; nerve growth factor expression; phospholipaseA,; lipoxygenase; immune-mediated glomerulonephritis

GROWTH FACTOR (NGF) regulates the development and maintenance of peripheral sympathetic and neural crest-derived sensory neurons (17). Recent data suggest that NGF also acts in the central nervous system as a trophic agent for the cholinergic neurons of the basal forebrain and of the caudate putamen (28). However, only little is known about the molecular mechanisms of NGF and NGF-receptor expression. Inflammatory mediators, in particular interleukin-1 (IL-l), tumor necrosis

factor-a (TNF-cw), and transforming growth factor-p (TGF-P), have been described as potent inducers of NGF synthesis in peripheral tissues and brain (9, 18, 19, 33). In addition to its neurotrophic action, NGF appears to exert potent biological effects on nonneuronal cells (34). There is increasing evidence that the NGF and NGFreceptor system may modulate inflammatory and immune reactions (24,32). High amounts of NGF are found at sites of inflammation and in inflammatory exudates (46). Furthermore, functional NGF binding sites have been detected on mononuclear lymphocytes and monocytes, and NGF was shown to induce growth and differentiation of human B cells (34). Renal mesangial cells not only participate in the regulation of glomerular filtration rate by their smooth muscle activity in response to vasoactive agents but are also considered to play a central role in the pathogenesis of immune-mediated glomerulonephritis (26, 36, 39). Furthermore, they have been shown to release a number of cytokines in vitro, including IL-l (21), IL-6 (13), TNFCY(2), platelet-derived growth factor (PDGF) (41), colony-stimulating factor I (CSF I) (29), granulocyte-macrophage CSF (4), and possibly also TGF-P1 (3). On the other hand, mesangial cells respond in a synergistic fashion to IL-l and TNF-a: by prostaglandin E, (PGE2) synthesis and phospholipase A2 (PLAZ) release (37), to IL-l, IL-6, and PDGF by cell proliferation (13, 21, 41), to IL-l by the release of a specific type IV collagenase (23), and to interferon-y (IFN-y) by upregulation of class II major histocompatibility antigen (25). Moreover, they express functional binding sites for CSF I (29) and TGFp (22). Thus it is tempting to speculate that the mesangium plays an important role in coordinating local immune functions. In the present study, we report that ILlp and TNF-cr selectively stimulate mesangial cells to synthesize and secrete biologically active NGF.

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Preparation of mesangial cell cultures. Renal glomeruli from male Sprague-Dawley rats (70-100 g body wt) were prepared under sterile conditions by a sieving technique and maintained in an in vitro system as described previously (35). The outgrowing cells were subcultured after treatment with 0.025 g/d1 trypsin 21 days after the first

0 1991 the American

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inoculation of the glomeruli. In a second step, single cells were cloned by limited dilution with 96-microwell plates. Clones with apparent mesangial cell morphology were used for further processing. The cells exhibited the typical stellate morphology. Moreover, there was positive staining with fluorescencelabeled antibodies for the intermediate filaments desmin and vimentin, considered to be specific for myogenic cells (45), and negative staining for factor VIII-related antigen (present on endothelial cells) and cytokeratin. Thus the possibility of a contamination with endothelial and epithelial cells or fibroblasts can be excluded. Furthermore, generation of inositol trisphosphate in response to angiotensin II (35) was used as a functional criterion for characterizing the cloned cell line. Cloned mesangial cells were grown at 37°C in RPM1 1640 cell culture medium, supplemented with 20% fetal calf serum (FCS), 100 IU/ml penicillin, 100 pg/ml streptomycin (all from Boehringer Mannheim, Mannheim, FRG), and 5 pg/ml bovine insulin (Sigma Chemical), in 75cm2 cell culture flasks in a humidified incubator containing 5% COZ. For stimulation, confluent mesangial cells cultured in 16-mm-diameter dishes were washed once and incubated with 1 ml of serum-free Dulbecco’s modified Eagle’s medium (Boehringer Mannheim) supplemented with fatty acid-free bovine serum albumin (0.1 mg/ml) (Sigma) with or without the different agents. After the appropriate incubation periods, the medium was withdrawn and immediately frozen in liquid nitrogen and stored at -70°C until assay of immunoreactive NGF as well as biologically active NGF in the PC12 bioassay. For RNA extraction, loo-mm culture dishes were used. After removal of the medium, cell lysis buffer (6) was given directly into the dishes and cellular RNA was extracted as described below. Passages 9-34 were used for the experimental work. Recombinant human IL-l& indomethacin, diclofenac, and staurosporine were prepared by Ciba-Geigy, Basel, Switzerland. Recombinant human IL-la was obtained from Biogen, Geneva, Switzerland. Recombinant human TNF-a was purchased from Boehringer Mannheim, and mepacrine and dexamethasone were from Sigma. Enzyme immunoassay for NGF. A two-site enzymelinked immunosorbent assay (ELISA) was used to measure immunoreactive NGF released into the culture medium (46). Briefly, polystyrene 96-well microtiter immunoplates (Nunc, Copenhagen, Denmark) were coated with polyclonal goat anti-NGF antibodies or with nonimmune goat serum. All incubations were performed in a moist chamber, and after each incubation step the plates were washed four times with phosphate-buffered saline (PBS) and 0.05% Tween 20. After an S-h coating step at 4°C nonspecific binding was blocked with a mixture of PBS, 0.05% Tween 20, and 1% FCS for 2 h at 2O’C. One hundred microliters of the cell culture medium were added to each well for 12 h at 4”C, whereby each plate contained a complete standard curve ranging from 0 to 100 pg/O.l ml of isolated NGF. Six micrograms of monoclonal rat antibody (clone 23~4) per well were added and incubated for 9 h at 4°C. Biotinylated goat immunoglobulins (1:8,000 dilution; Zymed) were incubated overnight at 4°C. and the procedure was continued

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by adding peroxidase-conjugated streptavidin (Zymed) at a 1:5,000 dilution for 3 h at 4°C. Immune sandwiches were detected by the addition of o-phenylenediamine (4 mg/plate; Merck, Darmstadt, FRG) in a freshly prepared solution containing 5 ~1 of 30% Hz02 in 10 ml substrate buffer. The reaction proceeded for 7-10 min at 20°C and was stopped with H2S04. Optical density was measured at 490 nm by means of a Dynatech MR 700 microplate reader. Absorbance of samples and standards was corrected for nonspecific binding (i.e., the absorbance in wells coated with normal goat serum). The NGF content in the samples was determined in relation to a NGF standard curve. Data are not corrected for recovery of NGF from the samples, which was routinely 70-80%, and were accepted only when values were greater than two standard deviations above the blank. Using this criterion, the limit of sensitivity of the NGF ELISA averaged 1 pg/ assay. Cytokines and inhibitory agents at the concentration used did not interfere with specific NGF content measurement. Statistical analysis was by analysis of variance (ANOVA), and the values are means t SE. RNA extraction and blot hybridization. Total RNA was extracted from mesangial cells in guanidinium hydrochloride and isolated according to a modified procedure of Cheley and Anderson (6). Contents of total RNA were determined by measurements of optical density at 260 nm. RNA was separated on denaturing 1.5% formaldehyde-agarose gels and subsequently transferred to nylon membranes by Northern blotting. Filters were hybridized to a 32P-labeled NGF mRNA complementary to the message sense that was obtained by in vitro transcription of a NGF cDNA clone encoding the precursor of mouse ,&NGF (40). The clone had been inserted into a SP6 plasmid. Unspecific signals were removed by treating filters with ribonuclease A. For quantitation, autoradiograms of the filters were scanned densitometrically, and the intensities of the signals were compared with known amounts of coelectrophoresed NGF message sense RNA. In general, NGF mRNA contents were calculated as femtograms per microgram total RNA and expressed as percent of controls (100%). Signals were normalized to @actin mRNA. Statistical analysis was by ANOVA (means t SE). RESULTS

Effects of cytokines on NGF synthesis in rat glomerular mesangial cells. A cell culture system of pure mesangial

cells from rat renal glomeruli has been established. Unstimulated cells (referred as control) produced detectable amounts of immunoreactive NGF. This basal NGF release amounted to 29.3 t 10.5 pg NGF/ml cell culture medium within 24 h and was observed throughout all experiments. As shown in Fig. 1, the incubation of the cells with the human recombinant cytokines IL-la, ILlp, or TNF-cw at a concentration of 1 nM each triggered only a weak stimulatory effect on NGF production (1.4to X-fold) after 24 h. However, the simultaneous addition of IL-lp and TNF-cw (both 1 nM) elicited a marked 13-fold increase of NGF released into the culture medium within 24 h. This svnergistic induction (P < 0.001) of

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400 T

n

.

0

z E s

rEl il-

Q F A

u t

u sl+ U

u i +

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1. Effects of cytokines on nerve growth factor (NGF) production in rat mesangial cells. Mesangial cells were incubated in presence of indicated cytokines at a concentration of 1 nM each, alone or in combination. After a 24-h incubation period, medium was removed and NGF was measured as described in MATERIALS AND METHODS. Data are means (error bars are *SE) of 4 independent experiments each determined at least in triplicate. Statistical analysis was by ANOVA analysis (* P < 0.05 and ** P < 0.001 compared with unstimulated controls). Synergistic effect of interleukin- lp (IL-lp) + tumor necrosis factor-a (TNF-cu) was demonstrated by comparison of the arithmetic sum of individual effects of IL-lp and TNF-cw after subtraction of control value with experimental value found for combined effect (ILl@ + TNF-CY) minus control value by Student’s t test (P < 0.001). FIG.

10”’

lo-'0 Cytokine

1o'g (M)

1o-8

FIG. 2. Dose-dependent NGF synthesis in mesangial cells induced by cytokines. Mesangial cells were incubated with indicated concentrations of IL-l@ (0), TNF-ar (0), or IL-lp + TNF-cu (0). Experiment with combination of cytokines (0) was performed at a constant concentration of TNF-a! (1 nM), whereas various concentrations of IL-l@ were used. After a 24-h incubation period, medium was removed and NGF was measured as described in MATERIALS AND METHODS. Data are means in % of control (error bars are *SE) of 4 independent experiments each determined at least in triplicate. Unstimulated cells (control) produced 21.2 t 3.7 pg NGF/ml within 24 h. * P < 0.05, ** P < 0.05 (only IL-l@-stimulated cells), and tt P c 0.001 compared with unstimulated controls. *** P < 0.001 compared with TNF-a! (1 nM)stimulated cells.

NGF synthesis revealed a remarkable specificity, because it was not initiated with IL-la in combination with TNFCY.

To determine whether cytokine-induced NGF release is a dose-dependent phenomenon, mesangial cells were incubated with various concentrations of IL-l@ and TNF-a), alone and in combination. The effects of IL-l& as well as of TNF-cu alone, were concentration dependent, with a first significant (P c 0.01) stimulation of NGF release occurring for both at 1 nM (Fig. 2). Moreover, we found that the effect on NGF synthesis induced by combined addition of IL-l@ + TNF-cu was strictly dependent on the IL-l@ concentration when TNF-cu remained constant at 1 nM. A maximal synergistic stimulation of NGF was detected at 1 nM IL-lp @O-fold). Higher concentrations of IL-l@ did not elicit a more pronounced specific effect. Unstimulated mesangial cells released small amounts of NGF gradually over time (Fig. 3, open circles). A first significant (P < 0.001) stimulation of NGF release in the presence of IL-lp + TNF-cu (both 1 nM) was reached after a 16-h incubation period, as shown in Fig. 3. The cytokines alone failed to significantly alter the NGF signal. To investigate whether the stimulation of NGF release is due to increased NGF synthesis, NGF mRNA levels in controls and cytokine-treated mesangial cells were quantified at various time points. As shown in Fig. 4 (A and B), NGF mRNA levels of mesangial cells stimulated with IL-l@ + TNF-cr began to increase after 4 h, attaining a maximal increase (&fold) by 8 h. Furthermore, NGF mRNA remained elevated for ~24 h.

Incubation

time

(h)

3. Time course of NGF released by cytokine-stimulated mesangial cells. Mesangial cells were incubated for indicated time periods with IL-l@ (o), TN& (A), or IL-lp + TNF-a! combination (;> (all 1 nM). Controls (0) are values in absence of cytokines. After indicated incubation periods, medium was removed anb NGF was measured as described in MATERIALS AND METHODS. Data are means in % of control (error bars are +SE) of 4 independent experiments each determined at least in triplicate. Unstimulated cells (control) produced 94.1 t 3.1 pg NGF/ml within a 48-h culture period. *P c 0.001 compared with unstimulated controls. FIG.

Thus these findings strongly suggest that stimulated NGF release is the result of enhanced NGF gene expression. The biological activity of the secreted NGF was tested by means of a PC12 cell bioassay system (11). We found that neurite outgrowth induced by the conditioned medium of mesangial cells that were stimulated with IL-16 + TNF-cu was completely inhibited by addition of NGF antibodies (data not shown). Thus our results provide evidence that mesangial cells indeed release biologically active NGF. Mechanisms involved in regulation of cytokine-induced NGF expression in mesangial cells. The mesangial cells

were incubated with optimal

cytokine concentrations

and

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1.3 kb

s - 300I =

200-

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'00

E

*

FIG. 5. Effects of phospholipase At inhibitors on cytokine-induied NGF synthesis in mesangial cells. Mesangial cells were stimulated simultaneously with IL-1B + TNF-rw (both 1 nM) in presence or absence of 200 PM mepacrine (A) or 1 PM dexamethasone (B). After a 24-h incubation period, medium was removed and NGF was measured as described in MATERIALS AND METHODS. Data are means in % of control (error bars are &SE) of 4 independent experiments each determined at least in triplicate. Unstimulated cells (control) produced 10.3 rt 2.5 pg NGF/ml within 24 h. *P < 0.05 and **P < 0.001 compared with appropriate controls (incubation without inhibitors).

2000 T

0 -2-

-4-

-8-

-24-

Time (h) 4. Effects of cytokines on NGF mRNA levels in mesangial cells. Mesangial cells were incubated for various time periods with combination of IL-lp + TNF-a (both 1 nM) or without cvtokines. A: autoradiogram of a representative experiment of Northern blot analysis of NGF mRNA (1.3 kb). Mesaneial cells were incubated for 24 h in absence (lanes 1-3) or presence (lanes 4-6) of cytokines. Total RNA of 5-10 pg was loaded onto gels. B: time course of NGF mRNA levels in unstimulated (open bars) or stimulated (solid bars) mesangial cells. Total cellular RNA was extracted and analyzed by quantitative Northern blotting as described in MATERIALS AND METHODS. Data (in B) are means (error bars are *SE) in % of control (n = 4). Average NGF mRNA content of controls amounted to 49 f 16 fg/pg total RNA. * P < 0.01 compared with unstimulated cells. FIG.

various enzyme inhibitors to study the regulation of NGF gene expression. Recent findings revealed that cytokines induce the release in mesangial cells of PLAz (37), one of the rate-limiting enzymes for arachidonic acid release (15). Therefore it was of interest to investigate the regulatory role of PLA2 on NGF synthesis by use of mepacrine (20), a potent PLA, inhibitor, and the synthetic glucocorticoid dexamethasone, which has been shown to inhibit PLAz secretion and eicosanoid synthesis in cytokine-stimulated mesangial cells (38). As shown in Fig. 5 (A and B), NGF production induced by IL-10 + TNFcy,as well as basal NGF release, was completely abolished by simultaneous addition of either mepacrine or dexamethasone. At the concentrations used, the inhibitors did not affect mesangial cell viability as monitored by a sensitive dye exclusion test (30) (data not shown). In conclusion, our findings suggest that PLAz may be in-

+

+

-+

6. Effects of cyclooxygenase inhibitors on cytokine-induced NGF production in mesangial cells. Mesangial cells were incubated in presence of indicated cytokines (1 nM) and cyclooxygenase inhibitors indomethacin (500 nM) or diclofenac (200 nM). After a 24-h incubation period, medium was removed and NGF was measured as described in MATERIALS AND METHODS. Data are means in % of control (error bars are &SE) of 4 independent experiments each determined at least in triplicate. Unstimulated cells (control) produced 26.6 z!z 1.3 pg NGF/ ml within 24 h. None of groups (incubated with inhibitors) differed significantly (P < 0.05) from appropriate controls. FIG.

volved in cytokine-stimulated NGF synthesis. In addition, enhanced PGE2 synthesis after cytokine treatment has been reported in mesangial cells (37). Thus it was of interest to study whether prostaglandin biosynthesis is a prerequisite for cytokine-stimulated NGF production. As shown in Fig. 6, exposure of the cells to IL-l@ and TNF-a( (alone or together) in the presence of

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the potent cyclooxygenase inhibitors indomethacin and diclofenac failed to modify the cytokine-induced NGF expression. Previous studies demonstrated that these agents, at the concentrations used, completely inhibited cyclooxygenase without affecting PLA2 activity (38). Thus these results clearly indicate that prostaglandins do not contribute to the mechanisms of NGF induction. In contrast, pretreatment with nordihydroguaiaretic acid (NDGA), which inhibits lipoxygenase activity (7), dose-dependently suppressed the induction of NGF by cytokines (half-maximal inhibitory concentration of -6 PM), suggesting that a lipoxygenase metabolite produced in response to cytokine stimulation mediates NGF expression (Table 1). Furthermore, to clarify whether an intact phospholipase C/protein kinase C (PKC) pathway is involved in NGF synthesis, cytokine-stimulated mesangial cells were exposed to staurosporine, a PKC inhibitor (43). Interestingly, staurosporine potentiated cytokine-induced NGF production in mesangial cells approximately twofold (Fig. 7), suggesting that stimulation of NGF production by IL-l@ + TNF-cr together is modulated by PKC. DISCUSSION

In the present study rat glomerular mesangial cells known to be involved in immune reactions also synthesize and release NGF. Mesangial cells are considered to play a crucial role in acute glomerular injury and nephritis. They respond to monocyte-derived cytokines by a rapid formation of potent proinflammatory mediators, e.g., interleukins, prostaglandins, and neutral protease, and express functional binding sites for IL-l, IL-6, TNFa, PDGF, IFN-7, CSF I, and TGF-P. The results presented in this report clearly indicate that IL-l@ and TNF-cu together are potent inducers of NGF synthesis in mesangial cells. This interesting observation leads to a series of questions. For example, how is NGF expression regulated and what is the physiological/pathophysiological role of NGF-producing cells in renal glomeruli with respect to immune processes? Neuronal lesion models have recently been used to investigate the regulation of NGF biosynthesis. It has been demonstrated that an increase in NGF and NGF 1. Effects of NDGA on cytokine-induced NGF synthesis in mesangial cells TABLE

NGF,

Treatment

Control IL-10 (1 nM) + NDGA + NDGA + NDGA + NDGA

+ TNF-cu (ml PM) (-5 ,uM) (-25 ,uM) (-100 PM)

(1 nM)

% of control

lOO& 14.8 1,156+61.1* 935t75.9 681+27.8f 169*27.8$ 102*53.7$

Values are means & SE in % of control; n = 4 experiments. Mesangial cells were incubated for 24 h in presence of indicated cytokines. Lipoxygenase inhibitor was added simultaneously. After incubation period, nerve growth factor (NGF) was measured as described in MATERIALS AND METHODS. Control mesangial cells produced 5.4 * 0.8 pg NGF/ml within 24 h. IL, interleukin; TNF, tumor necrosis factor; NDGA, nordihydroguaiuretic acid. * P < 0.001, compared with unstimulated cells. t P < 0.05 and $ P < 0.001, compared with cytokinetreated cells.

FIG. 7. Effects of staurosporine on cytokine-induced NGF expression in mesangial cells. Mesangial cells were incubated with IL-lp + TNF-ar (both 1 nM) in presence or absence of staurosporine (200 nM). After a 24-h incubation period, medium was removed and NGF was measured as described in MATERIALS AND METHODS. Data are means in % of control (error bars are &SE) of 4 independent experiments each determined at least in triplicate. Unstimulated cells (control) produced 10.3 t 2.5 pg NGF/ml within 24 h. * P < 0.001 compared with appropriate controls (incubation without inhibitor).

mRNA at sites of injury in the peripheral nervous system is mediated by compounds released by infiltrating monocytes. Furthermore, IL-l, one of the most effective inflammatory mediators, appears to be responsible for the potent stimulation of NGF synthesis in cultured fibroblasts (19). Recent data strongly suggest that IL-l induces NGF production also in brain (33). Moreover, ILl@ and TNF-a synergistically stimulate NGF release from cultured astrocytes (9). The present findings provide evidence that this regulatory mechanism is not restricted to the nervous system but also occurs in other cells associated with immune function. In mesangial cells, a stimulatory effect on NGF synthesis was initiated by simultaneous addition of IL-l@ and TNF-cu. If added together, IL-lp and TNF- CYelicited a marked induction of NGF gene expression. This synergistic effect was dose dependent and maximal at 1 nM. Interestingly, IL-la! in combination with TNF-CY failed to potentiate NGF production. Differences in the pattern of biological activities of IL-la and IL-l@ alone have been reported previously (10, 37). In several biological systems IL-l and TNF-cu act in a synergistic way (9,16,37); but, to our knowledge, this is the first report providing evidence for a discrimination between the a- and ,&forms of IL-l when applied with TNF-a to induce an enhanced response. Moreover, the biological activity of the IL-la used in this study has been tested previously in terms of PGEz synthesis and PLA2 secretion from mesangial cells (37). Our studies showing that cytokine treatment of mesangial cells produced a marked increase in NGF mRNA levels (maximal effect was S-fold within 8 h) strongly

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suggest that increased NGF release is the result of elevated NGF gene expression. However, the maximal induction of NGF mRNA after 8 h may be due to an indirect effect. For example, it is well established that cross induction of IL-l and TNF-a occurs in many biological systems (16). Furthermore, IL-l and TNF-a are potent inducers of other cytokines including IL-6 (l), which acts as an autocrine growth factor for mesangial cells in vitro (13). Interestingly, IL-6 transgenic mice develop mesangioproliferative glomerulonephritis (42). In addition, IL-6 has been demonstrated to stimulate NGF release by cultured astrocytes (8). However, it is unclear whether IL-6 is involved in IL-lp- and TNF-cuinduced NGF synthesis. The signal transduction mechanisms and intracellular molecular events by which IL-l and TNF-a exert their effects on target cells are not defined (27, 31). However, the fact that dexamethasone as well as mepacrine abolish IL-l@ and TNF- a-induced NGF expression in mesangial cells provide strong evidence that cytokine-mediated PLA2 activation in mesangial cells may trigger NGF expression and secretion. In contrast, indomethacin and diclofenac at concentrations completely inhibiting cyclooxygenase failed to modify cytokine-stimulated NGF synthesis, clearly indicating that prostaglandins do not contribute to cytokine-induced NGF production. The fact that NDGA dose-dependently inhibited cytokineinduced NGF production indicates that lipoxygenase metabolites are involved in NGF gene expression. In this context it is noteworthy that lipoxygenase inhibitors such as NDGA abolish the induction of c-fos by TNF in the adipogenic TA 1 cell line (12). Because it is known that TNF stimulates platelet-activating factor (PAF) in mesangial cells (5), we addressed the question of whether PAF is able to modulate NGF production. Neither PAF or specific PAF-receptor antagonists had any significant influence on cytokine-stimulated NGF synthesis excluding a mediator role of PAF. Our experiments with the PKC inhibitor staurosporine suggest that PKC modulates cytokine-mediated signal transduction. These results are in agreement with a finding showing that PKC is involved in inactivation of the IL-l receptor on a human transformed B cell line (14). Moreover, staurosporine has been reported to amplify IL-l- and TNF-ainduced increases in PGEz formation in dermal fibroblasts (44). The pathogenesis of immune-mediated glomerulonephritis is not completely understood, but one of the crucial events involved in this disease appears to be an immunological injury to mesangial cells (3). Inflammatory cytokines including IL-l and TNF-cr accumulate at sites of injury within the glomeruli and are probably released by activated monocytes. As a consequence, IL1 and TNF-cu trigger a variety of cellular responses. The present study reports for the first time that rat glomerular mesangial cells express biologically active NGF on cytokine stimulation, which may act as a signaling protein between resident glomerular cells and invading lymphocytes. Recent findings demonstrate that functional NGF receptors are present on human monocytes and lymphocytes (34). Because these cells are involved in immune reactions, NGF may modulate local immune

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responses within the glomeruli. To exclude that synthesis of NGF by mesangial cells is a new characteristic of differentiation of these cells in culture, RNA expression of NGF was measured in freshly isolated rat glomeruli. We observed that isolated glomeruli synthesize NGF mRNA, as monitored by polymerase chain reaction (C. Boeckh, J. Pfeilschifter, and U. Otten, unpublished observations), clearly documenting the in vivo relevance of our data. It is of considerable interest to check whether mesangial cells express functional NGF receptors. In that case, NGF could act in an autocatalytic fashion. Furthermore, a cytokine network including NGF may play a role in the pathophysiology of renal diseases. A better characterization of the NGF and NGF-receptor system of human mesangial cells is necessary to further elucidate the physiological and pathophysiological role of NGF. We acknowledge the skillful technical assistance of Barbara Thuring and thank Karin Cron for help with PC12 cell cultures. This study was supported by Swiss National Foundation for Scientific Research Grants 31-25690.88 and 31-29954.90 and by Deutsche Forschungsgemeinschaft SFB 325. Address for reprint requests: U. Otten, Dept. of Physiology, Univ. of Basel, Vesalianum, Vesalgasse 1, CH-4051 Base& Switzerland. Received 28 December 1990; accepted in final form 26 June 1991. REFERENCES 1. AKIRA,

T. TAGA, AND T. KISHIMOTO. Biology of cytokines: IL 6 and related molecules (IL 1 and

S., T. HIRANO,

multifunctional TNF). FASEB

J. 4: 2860-2867,199O. 2. BAUD, L., J.-P. OUDINET, M. BENS, L. NOE, RONDEAU, J. ETIENNE, AND R. ARDAILLOU.

M.-N.

PERALDI,

E.

Production of tumor necrosis factor by rat mesangial cells in response to bacterial lipopolysaccharide. Kidney ht. 35: 1111-1118, 1989. 3. BORDER, W. A., S. OKUDA, L. R. LANGUINO, M. B. SPORN, AND E. RUOSLAHTI. Suppression of experimental glomerulonephritis by antiserum against transforming growth factor pl. Nature Lond. 346: 371-374,199O. 4. BUDDE, K., D. L. COLEMAN,

J. LACY, AND R. B. STERZEL. Rat mesangial cells produce granulocyte-macrophage colony-stimulating factor. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26):

F1065-F1078,1989. 5. CAMUSSI, G., E. TURELLO, BAGLIONI. Tumor necrosis

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