Recent reports have focused attention on the relationship between inflammatory nephropathies and chemokines (1,2), a family of small proinflammatory peptides that are primarily known because of their leukocyte chemoattractant activity (3,4). There are at least four families of chemokines, but only two have been extensively characterized (3,4). In general, CXC chemokines attract neutrophils, whereas the chemokines belonging to the CC family act primarily on monocytes, although they can also attract lymphocytes, basophils, and eosinophils (5). A new classification system for the different families of chemokines was recently proposed (6). The activities of chemokines are not limited to chemotaxis and involve cells other than leukocytes (4). For example, increased expression of the interferon-γ (IFN-γ)-inducible protein of 10 kD (IP-10) and the monokine induced by IFN-γ (Mig), now named CXCL10 and CXCL9, respectively (6), has been observed in a variety of tissues affected by inflammatory conditions, including murine models of glomerulonephritis (GN) (1,2,7–9). In addition, IP-10 was observed to be produced by mesangial cells, and the kidney seems to be the most important source of IP-10 in response to IFN-γ stimulation (10).
A receptor for both IP-10 and Mig that mediates Ca2+ mobilization and chemotaxis in activated lymphocytes, i.e., the CXC chemokine receptor 3 (CXCR3), has been characterized and cloned (11). We recently reported high levels of expression of CXCR3 in mesangial cells from patients with proliferative GN (12). Moreover, CXCR3 was also observed on the surface of cultured human mesangial cells (HMC) and seemed to mediate both intracellular Ca2+ influx and cell proliferation (12). Therefore, among patients with proliferative GN, the chemokines IP-10 and/or Mig not only may be responsible for the attraction of infiltrating mononuclear cells into the inflamed tissue but also may directly stimulate the proliferation of mesangial cells. More recently, it was observed that IP-10 and Mig also induce chemotaxis and extracellular signal receptor-activated kinase activation in HMC (13). However, the expression of IP-10 and Mig in human GN has not yet been demonstrated, and the mechanisms that modulate the secretion of CXCR3-binding chemokines at the kidney level are only partially understood.
In this study, we demonstrate that both IP-10 and Mig mRNA and protein are widely expressed in the kidneys of patients with proliferative GN, where they are mainly localized to resident glomerular cells, especially at the level of mesangial cells. Moreover, primary cultures of both HMC and human visceral epithelial cells (HVEC) synthesized IP-10 and Mig after activation with IFN-γ, and even more in response to IFN-γ plus tumor necrosis factor-α (TNF-α). More importantly, at least in this in vitro system, IP-10 and Mig production stimulated by IFN-γ plus TNF-α seemed to be strongly reduced by the addition of nitric oxide (NO) donors, through a cGMP-independent, NF-κB-dependent pathway, suggesting that an inhibitory effect on the production of these chemokines may account at least in part for the protective effects of NO that were previously observed in some experimental models of proliferative GN.
Materials and Methods
Renal biopsy specimens from a total of 26 subjects were used throughout the study. Eleven renal biopsies were obtained from patients with proliferative glomerulopathies (five with membranoproliferative GN, three with IgA nephropathies, two with crescentic GN, and one with focal segmental glomerulosclerosis with mesangial cell proliferation), which were selected because of the extensive degree of mesangial cell increases. Ten renal biopsies were obtained from patients with nonproliferative GN (six with membranous GN, one with focal glomerular sclerosis, two with angiosclerosis, and one with amyloidosis), exhibiting no or very low levels of mesangial cell proliferation. Control specimens were obtained from the normal kidney tissue of five patients who were undergoing nephrectomy for the treatment of primary localized renal tumors. The procedures used in this study were in accordance with the guidelines of the regional ethics committee on human experimentation.
Cloning and Sequencing of the IP-10 and Mig Probes
IP-10 and Mig cDNA probes were prepared as described previously (14).
In Situ Hybridization
In situ hybridization was performed according to a previously described technique (14). Exposure times varied from 15 to 20 d. Negative control samples consisted of sections hybridized with a sense RNA probe.
Immunohistochemical analyses were performed with 10-μm cryostat sections, as described (12), by using the avidin-biotin peroxidase method (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) and rabbit anti-human IP-10 or Mig polyclonal antibodies (Ab) (Peprotech, London, UK). 3-Amino-9-ethylcarbazole (AEC) (Sigma Chemical Co., St. Louis, MO) was used as a peroxidase substrate. Sections were counterstained with Gill’s hematoxylin (Merck, Darmstadt, Germany). In negative control experiments, the primary Ab was omitted or replaced with mouse ascites fluid.
In some experiments, IP-10 or Mig and α-smooth muscle actin (α-SMA), von Willebrand factor, CD35, CD68, CD3, CD20, or CD56 were colocalized on the same sections in double-label immunohistochemical analyses, according to a method detailed elsewhere (12). Briefly, the anti-IP-10 or anti-Mig Ab were applied first, and AEC (red color) was used as a peroxidase substrate. Sections were subsequently exposed to anti-α-SMA monoclonal Ab (mAb) (clone 1A4; Sigma), anti-von Willebrand factor Ab (rabbit anti-human von Willebrand factor polyclonal Ab; Dako, Glostrup, Denmark), anti-CD35 mAb (Pharmingen, San Diego, CA), anti-CD68 (clone EBM11; Dako), anti-CD3 (clone UCHT-1; Cymbus Biotechnology, Chandlers Ford, UK), anti-CD20 (clone L26; Dako), or anti-CD56 mAb (clone MOC-1; Dako), and Vector SG (bluish-gray color; Vector Laboratories) was used as a chromogen. No counterstain was applied. Proliferating cell nuclear antigen (PCNA) and α-SMA were colocalized on the same sections in double-label immunohistochemical analyses, using formalin-fixed, paraffin-embedded portions of the same specimens as used for the study. Anti-PCNA mAb (clone PC10; Dako) was applied first, and Vector SG (bluish-gray color; Vector Laboratories) was used as a peroxidase substrate. Sections were subsequently exposed to anti-α-SMA Ab, and AEC (red color) was used as a chromogen. No counterstain was applied.
Cultures of HMC and HVEC were obtained from macroscopically normal renal tissue from patients with localized tumors who were undergoing nephrectomy, as described previously (12).
LightCycler Quantitative Reverse Transcription-PCR
DNA amplifications were performed by using the LightCycler quantitative reverse transcription (RT)-PCR (Idaho Technology, Idaho Falls, ID) (15). The IP-10 amplicon was a 239-bp sequence that was amplified by using the forward primer 5′-ATCAAACTGCGATTCTGATTTGCTGCCTTA and the reverse primer 5′-TGGCCTTCGATTCTGGATAG. The first hybridization probe was 3′-labeled with fluorescein [5′-CCTGTTAATCCAAGGTCTTTAGAAAAACTT(fluo)-3′], and the second probe was 5′-labeled with Cy5 [5′-AAATTATTCCTGCAAGCCAATTTTTGTCCA(P)-3′].
The Mig amplicon was a 183-bp sequence that was amplified by using the forward primer 5′-TGGTGTTCTTTTCCTCTTGGGCATCATCTT and the reverse primer 5′-GGGAAGGACGCTCTTTTAACTTTAGTAACG. The first hybridization probe was 3′-labeled with fluorescein [5′-GCTGGTTCTGATTGGAGTGCAAGGAACCCC(fluo)-3′], and the second probe was 5′-labeled with Cy5 [5′-GTAGTGAGAAAGGGTCGCTGTTCCTGAAT(P)-3′] (15).
The inducible NO synthase (iNOS) amplicon was a 168-bp sequence that was amplified by using the forward primer 5′-CCAGTCCAGTGACACAGGATGACCTTCAGT and the reverse primer 5′-TGCCCCAGTTTTTGATCCTCACATGCCGTG. The first hybridization probe was 3′-labeled with fluorescein [5′-CTCAGCAAGCAGCAGAATGAGTCCCCGCAG(fluo)-3′], and the second probe was 5′-labeled with Cy5 [5′-CCCTCGTGGAGACGGGAAAGAAGTCTCCAG(P)-3′].
The β-actin amplicon was a 229-bp sequence that was amplified by using the forward primer 5′-TGACGGGGTCACCCACACTGTGCCCATCT and the reverse primer 5′-TGGAAGCAGCCGTGGCCATCTCTTGCTCGA. The first hybridization probe was 3′-labeled with fluorescein [5′-CTGGCCGGGACCTGACTGACTACCTCATGA(fluo)-3′], and the second probe was 5′-labeled with Cy5 [5′-GATCCTCACCGAGCGCGGCTACAGCTTCAC(P)-3′]. Primers and probes were synthesized and purified by Genset (Paris, France). Amplification was performed with 40 cycles of denaturation (94°C, 0 s; ramp rate, 20°C/s), annealing (59°C, 15 s; ramp rate, 20°C/s), and extension (74°C, 0 s; ramp rate, 0.8°C/s). All IP-10 and Mig mRNA concentrations obtained for every sample were normalized to the β-actin content. The β-actin oligodeoxynucleotides were selective for β-actin mRNA and did not recognize DNA sequences (16).
Enzyme-Linked Immunosorbent Assays
The ability of HMC and HVEC to produce IP-10 and Mig was evaluated in cell-free supernatants by using a homemade enzyme-linked immunosorbent assay system, with two pairs of anti-IP-10 or anti-Mig mAb (Pharmingen). HMC and HVEC were grown to 70 to 80% confluence and then rendered quiescent by incubation for 48 h in serum-free medium. Medium was then replaced with serum-free medium containing recombinant IFN-γ (1000 U/ml; Endogen, Woburn, MA), TNF-α (8 ng/ml; Janssen Biochimica, Jachem, Belgium), or both, and cell-free supernatants were collected after 8, 16, and 24 h and analyzed for IP-10 and Mig contents. Antagonists of guanylate cyclase such as NS2028, Rp-8-(p-chlorophenylthio)-cGMP, and 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one, cGMP analogues such as 8-bromo-cGMP, and the iNOS inhibitor 1400W were purchased from Merck.
Electrophoretic Mobility Shift Assays
Nuclear extracts were prepared according to a modification of the method described by Dignam et al. (17). For binding reactions, 10 mg/ml levels of nuclear extracts were incubated with 5 μg of poly(dI-dC) (Pharmacia Biotech, Piscataway, NJ), in 30 μl of reaction mixture containing 20 mM Hepes (pH 7.9), 37.5 mM KCl, 0.2 mM ethylenediaminetetraacetate, 1.0 mM dithiothreitol, and 10% glycerol, for 10 min at room temperature. Oligonucleotide duplex probes (end-labeled with T4 polynucleotide kinase and [γ-32P]ATP at 5 × 104 cpm) were then added to the reaction mixtures, which were incubated for 20 min at room temperature. Reaction products were analyzed by nondenaturing electrophoresis in 6% polyacrylamide gels, with 0.5× TBE buffer (44.6 mM Tris, 44.4 mM borate, 0.5 mM ethylenediaminetetraacetate), at room temperature. Gels were dried and exposed to x-ray film at −70°C for autoradiography. For competition experiments, unlabeled oligonucleotides were added in molar equivalence or excess at room temperature for 15 min before the addition of the radiolabeled probe. Oligonucleotide probe sequences were taken from the report by Majumuder et al. (18).
Nitrite (NO2−) levels were determined by using a commercially available kit (Merck), with addition of the Griess reagent (19) to supernatants of stimulated HMC and HVEC. Absorbance was measured at 540 nm, and NO2− concentrations were determined by using sodium nitrite as a standard.
Statistical analyses were performed by using the t test for unpaired data. Results were considered significant at P <0.05.
Expression of IP-10 and Mig mRNA and Protein in Kidneys of Subjects with GN
The expression of mRNA for IP-10 and Mig in normal kidneys, as well as in kidneys of patients with different forms of GN, was assessed by using in situ hybridization. IP-10 and Mig mRNA expression was virtually absent in normal kidneys (Figure 1, A and B), whereas biopsy specimens from kidneys of patients with proliferative GN consistently demonstrated high levels of both IP-10 and Mig mRNA (Figure 1, C and D). Sections hybridized with a sense IP-10 or Mig RNA probe demonstrated virtually no signal (data not shown). In kidneys from patients with GN, a large number of glomeruli exhibited intense signal throughout the tuft. Furthermore, part of the signal was localized to inflammatory infiltrates (Figure 1, C and D).
When the expression of IP-10 and Mig proteins was assessed in the same specimens by using immunohistochemical analyses, virtually no staining was observed in normal kidneys (Figure 1, E and F). In contrast, strong immunoreactivity for IP-10 and Mig was detected in kidneys of patients with GN, particularly at the levels of both glomerular structures and infiltrating mononuclear cells (Figure 1, G and H). No staining was observed in the same tissues with omission of the primary Ab or replacement with an isotype-matched control Ab with irrelevant specificity (data not shown). Combined in situ hybridization assays for IP-10 and Mig mRNA and immunohistochemical analyses for α-SMA demonstrated that mesangial cells were the main producers of the two chemokines, among resident glomerular cells (Figure 2, A to D). Double-immunostaining for IP-10 or Mig and CD35, a marker of glomerular podocytes (Figure 2F), demonstrated that IP-10 was localized to visceral epithelial cells in only a few cases, whereas larger numbers of glomerular podocytes produced Mig (Figure 2G). Interestingly, IP-10 and Mig expression by resident glomerular cells was virtually a selective property of kidneys exhibiting proliferative GN, as also demonstrated by double-immunostaining for α-SMA and PCNA (Figure 2E and Table 1). Furthermore, different subtypes of infiltrating leukocytes producing IP-10 and Mig were identified by double-label immunohistochemical analyses for these chemokines and CD68 (monocytes/macrophages), CD3 (T cells), CD56 (natural killer cells), or CD20 (B cells); the great majority of infiltrating mononuclear cells producing IP-10 and/or Mig were monocytes/macrophages, as demonstrated by their staining for CD68 (Figure 2, H and I). The results of semiquantitative assessments of IP-10 and Mig mRNA expression in the kidney biopsy specimens examined are summarized in Table 1.
Expression of IP-10 and Mig mRNA and Protein by Primary Cultures of HMC and HVEC and Modulation by NO Donors
To provide additional data on IP-10 and Mig expression by HMC and HVEC, the production of these chemokines by unstimulated and stimulated primary cultures of both cell types was then assessed. Unstimulated HMC or HVEC produced neither IP-10 nor Mig, whereas stimulation with IFN-γ induced modest, but detectable, production of both IP-10 and Mig (Figure 3). The stimulatory effect of IFN-γ on IP-10 and Mig production by HMC and HVEC was remarkably increased by costimulation with TNF-α, whereas no effect was observed with TNF-α alone (Figure 3).
Because NO has been demonstrated to exert either protective or damaging effects on kidney inflammatory processes, depending on the different models of experimental GN or the time of NO administration (20–22), the possibility that NO might have effects on IP-10 and Mig production by HMC was investigated. To this end, the effects of the NO donor sodium nitroprusside (SNP) on IP-10 and Mig production after maximal stimulation of HMC and HVEC with IFN-γ plus TNF-α were assessed. SNP induced dose-dependent inhibition of both IP-10 and Mig production (Figure 4A), which became detectable after 4 h and peaked at 16 h (Figure 4B). By using LightCycler quantitative RT-PCR, a strong inhibitory effect of SNP on the expression of IP-10 and Mig mRNA by both HMC and HVEC was also observed (Figure 5). The same results were obtained by using another NO donor, such as S-nitroso-N-acetylpenicillamine (SNAP). These inhibitory effects were not dependent on a toxic activity of NO donors in HMC, because parallel cell cultures treated for the same periods of time with the same concentrations of SNP or SNAP did not exhibit any loss of cell viability, as indicated by their uptake of propidium iodide. Moreover, the possibility of a potential regulatory role of endogenously produced NO could be excluded, at least under our experimental conditions. First, nitrites in cellular supernatants were virtually undetectable in the absence of NO donors and reflected their concentrations when NO donors were added to the cultures. Second, iNOS mRNA expression, as assessed by using LightCycler quantitative RT-PCR in both HMC and HVEC, was absent or negligible. Finally and most importantly, the specific iNOS inhibitor 1400W did not exert any additional effect on IP-10 production induced by IFN-γ plus TNF-α (data not shown).
Evidence that Suppression by NO Donors of IP-10 Production Induced by IFN-γ plus TNF-α Is Attributable to cGMP-Independent Inhibition of NF-κB Activation
Because IFN-γ plus TNF-α induction of IP-10 transcription requires cooperation of the IFN-stimulated response element (ISRE) site with at least one of the two NF-κB sites in the IP-10 promoter (18), we investigated whether the inhibitory effect of NO donors was related to direct binding of transcription factors to the ISRE site or the NF-κB sites. Treatment of HMC with NO donors did not affect the amounts of IFN-γ-induced DNA-protein complexes associated with the ISRE oligonucleotide (data not shown), suggesting that NO donors did not directly inhibit transcription factor binding to the ISRE site. In contrast, in experiments performed with κB2 oligonucleotides, NO donors markedly decreased the amounts of shifted complexes induced by IFN-γ plus TNF-α (Figure 6A). Therefore, NO donors seem to directly inhibit NF-κB activation by acting at the κB2 site in the IP-10 promoter. This effect was specific, because it could be blocked by the addition of 40 ng of unlabeled ISRE or κB2 oligonucleotide (Figure 6A). In addition, the activation of soluble guanylyl cyclase by exogenous NO did not contribute to the observed decrease in cytokine-induced IP-10 and Mig expression, because a cGMP analogue (8-bromo-cGMP, 10 μM to 1 mM) did not inhibit IP-10 or Mig protein expression (Figure 6B) and guanylate cyclase antagonists [Rp-8-(p-chlorophenylthio)-cGMP or NS2028, 10 μM to 1 mM] did not reverse the SNP-mediated inhibition of IP-10 and Mig synthesis (Figure 6B and data not shown).
The immune system can mount distinct and selective responses according to the type of infectious or antigenic challenge. In the context of such heterogeneity, two extremely polarized forms of CD4+ T helper (Th) cell-mediated specific immune responses have been described, on the basis of their profiles of cytokine production (23). Th1 cells, which produce interleukin-2 (IL-2), IFN-γ, and TNF-β, activate macrophages and are responsible for cell-mediated immunity, preferentially develop during infections by intracellular bacteria, and are involved in the pathogenesis of organ-specific autoimmune disorders. In contrast, Th2 cells, which produce IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, induce strong Ab responses by B cells and induce eosinophil activation, predominate during infestations by gastrointestinal nematodes, and are responsible for allergic reactions (23–25). Evidence suggesting that severe proliferative GN and crescentic GN are characterized by Th1 cell-dominated nephritogenic immune responses has recently been accumulated (26–28). Furthermore, several findings suggest that chemokines may be crucial for the recruitment and/or homing of Th1 or Th2 cells in inflamed tissues, inasmuch as distinct chemokine receptors seem to be preferentially associated with one or the other T cell subset. Th1 cells usually express CXCR3 and CC chemokine receptor 5 (CCR5), whereas Th2 cells preferentially express CCR8, CCR4, and, in small amounts, CCR3 (29–32). It is of note, however, that chemokine receptors are also present on cell types other than leukocytes and that chemokines not only play essential roles in leukocyte trafficking and homing but also act as regulatory molecules in leukocyte maturation, angiogenesis, and hematopoiesis (4). Because of their pleiotropic functions, it was recently hypothesized that chemokines and their receptors could contribute to the generation of tissue inflammatory reactions by both favoring the recruitment and/or homing of different types of circulating leukocytes and exerting direct effects on resident cells (2–4). In agreement with this view, we recently demonstrated that the chemokine receptor CXCR3 is expressed by mesangial cells in the kidneys of patients affected by proliferative GN, as well as by primary cultures of HMC, and that its activation by CXCR3 ligands induces Ca2+ influx in and mediates cell proliferation of HMC (12). In addition, it was demonstrated that IP-10 and Mig induced HMC proliferation through sustained activation of the extracellular signal receptor-activated kinase pathway (14).
In this study, both mRNA and protein expression of IP-10 and Mig, two chemokines that are mainly induced by IFN-γ and share the ability to bind to CXCR3, was assessed in normal kidneys and in kidneys of patients with different forms of GN. As expected, IP-10 and Mig were not detectable in normal kidneys, but they were highly expressed in the kidneys of patients with proliferative GN. Surprisingly, although infiltrating inflammatory cells in biopsy specimens obtained from patients affected by nonproliferative GN also expressed IP-10 and Mig, double-immunostaining experiments demonstrated that the synthesis of these chemokines by resident glomerular cells was a property of proliferative GN, in agreement with their proposed roles in the control of mesangial cell survival, chemotaxis, and proliferation (12,13). The ability of mesangial cells to produce high levels of IP-10 and Mig was confirmed by the demonstration that primary cultures of HMC and HVEC released large amounts of these chemokines after combined stimulation with IFN-γ and TNF-α, as previously reported for other cell types (33). Taken together, these observations may account for at least some mechanisms involved in the pathogenesis of proliferative GN. It is reasonable to speculate that the initial event, such as deposition of immune complexes, interactions of IgA with Fc receptors expressed on mesangial cells, or other insults, may induce both IP-10 and Mig production by resident glomerular cells (34), thus favoring the transmigration of Th1 cells and macrophages through the activation of adhesion molecules. Accordingly, Th1 effectors have been observed in the glomeruli of patients affected by severe proliferative GN, as well as in the glomeruli of patients affected by active forms of IgA nephropathy (35). Activated Th1 cells and macrophages are able to produce IFN-γ and TNF-α, which in turn can amplify production of both IP-10 and Mig. Interestingly, whereas treatment of proliferating mesangial cell cultures with IFN-γ leads to reduced DNA synthesis (36,37), administration of IFN-γ before the stimulation of mesangial cells with platelet-derived growth factor or epidermal growth factor dramatically increases cell proliferation, suggesting a possible pathogenic role of Th1 cells recruited by glomerular expression of IP-10 and Mig in the early phase of proliferative GN. Whether the same initial insult that induces IP-10 and Mig production, and consequently the recruitment of Th1 cells and macrophages, is responsible for the upregulation of CXCR3 expression by mesangial cells in the same kidneys is presently unclear. However, the interactions of IP-10 and Mig with their receptors expressed by mesangial cells could account for chronic stimulation of mesangial cell proliferation; therefore, the production of these chemokines provides an explanation for multiple aspects of the inflammatory reaction characteristic of proliferative GN.
Another interesting observation from this study is the strongly inhibitory effect of NO donors, such as SNP and SNAP, on both IP-10 and Mig mRNA expression, as well as IP-10 and Mig protein production, by HMC. NO has been observed to exert many effects on renal function (18–20), and increased NO synthesis has been observed in different models of GN, such as rat nephrotoxic serum nephritis, Heymann nephritis, anti-Thy 1.1 nephritis, and the nephritis of MLR-lpr/lpr mice (33–37). Therefore, the inhibitory effects of NO donors on IP-10 and Mig production that were observed in this study may account for the inhibitory activity of NO on mesangial cell proliferation (38). A recent report demonstrated that the activation of IP-10 transcription by IFN-γ plus TNF-α occurs through cooperation of the ISRE site with the NF-κB sites in the IP-10 promoter (20). Accordingly, the mechanisms for the effects of NO were independent of cGMP production, occurred at the transcriptional level, and involved the inhibition of IFN-γ- plus TNF-α-stimulated NF-κB activity. These data are consistent with previous findings that NO decreased cytokine-induced vascular smooth muscle cell expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 via cGMP-independent inhibition of NF-κB activation (39) and that NO inhibited smooth muscle cell proliferation mainly via a cGMP-independent mechanism (40). More importantly, the results of this study might explain the different effects of NO in the evolution of some nephritis models, in which NO can be either protective or damaging, depending on the experimental model used or the time of NO administration (40–47). This suggests that, at least in some mesangioproliferative disorders, NO donors administered at the appropriate times might exert potentially beneficial effects.
The experiments reported here were supported by funds from the Associazione Italiana per la Ricerca sul Cancro, from the Azienda Ospedaliera of Florence, from the Ministero Della Sanita (Programma per la Ricerca Finalizzata 1999 and 2000), and from Ministero Universita E[Combining Acute Accent] Ricerca Scientifica.
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