expression in crescentic glomerulonephritisInterleukin-18 (IL-18), originally designated interferon gamma (IFN-
)–inducing factor (IGIF), is a cytokine that has pleiotropic effects on several types of cells, especially immune cells1. IL-18 strongly induces IFN- production, natural killer (NK) cell cytotoxicity, and T-cell proliferation1,2. It has also been demonstrated that IL-18 induces the production of other cytokines, such as interleukin-2 (IL-2) and granulocyte/macrophage-colony-stimulating factor (GM-CSF) by T cells, and decreases interleukin-10 (IL-10)2,3. IL-18 is involved in tumor necrosis factor (TNF)- and Fas-ligand-mediated endotoxin-induced liver injury4. In addition, IL-18 plays a role in antitumor immunity5. Increased IL-18 expression is associated with autoimmune diseases such as the model of nonobese diabetes in the mouse and, more recently, at the onset and during the course of active experimental autoimmune encephalomyelitis6,7. IL-18 has also been identified to induce proinflammatory and catabolic responses in articular chondrocytes of patients with rheumatoid arthritis8. These findings indicate that IL-18 plays a role in various immunologic events in inflammatory, neoplastic, and autoimmune diseases.
IL-18 is synthesized as a polypeptide precursor (pro-IL-18) with no putative hydrophobic signal peptide. The precursor of IL-18 is cleaved after Asp35-X. The interleukin-1
(IL-1) converting enzyme (ICE) cleaves proIL-18 at the authentic processing site, activating IL-18 and facilitating its export, as has been demonstrated for human and mouse pro-IL-186,7,9,10. The amino acid sequence of IL-18 includes the IL-1 signature–like sequence and presents 15% homology at the amino acid level with IL-1 protein. Despite their similarities, IL-18 and IL-1 exhibit different biologic activities, transmitted through their specific receptors8.
IFN- is a cytokine associated with a variety of infectious, inflammatory, and autoimmune diseases11. IFN- is produced by activated CD4+ T (Th1), CD8+ T, or NK cells, and promotes the activation of macrophages (M
) and NK cells, as well as contributes to the regulation of T-helper cell immune responses12. IFN-, TNF, and cytokine products of activated M, such as IL-1, are implicated in the pathogenesis of immune complex–mediated glomerular injury13,14. Furthermore, IFN- is essential for the development of glomerulonephritis (GN) in (NZBXNZW) F1 and MRL-Faslpr lupus mice. Studies in MRL-Faslpr mice have suggested that IFN- receptor signaling is essential not only for the initiation but also for the acceleration and apoptotic destruction of the autoimmune kidney15.
The severe anti-glomerular basement membrane (GBM) antibody (Ab)-induced crescentic GN of WKY rats is a CD8+-dependent and M-related GN16. It is characterized by glomerular infiltration of many cell types that are able to produce INF-, including CD4+, CD8+, and NK cells. These cells also have been demonstrated to express IL-18 receptor (IL-18R). Accordingly, we investigated the involvement of IL-18 in the expression of IFN- in this model of crescentic GN. We found a rat IL-18 5'-untranslated region (UTR) translational inhibitor that was responsible for restricted production of IL-18. This 5'-UTR translational inhibitor was expressed in anti-GBM GN and correlated with a low IFN- mRNA and protein expression in this model of GN. The treatment with recombinant, active IL-18 of WKY with anti-GBM GN induced an increase of glomerular IFN- levels and worsening of the GN by increasing CD8+ cells and M. These data suggest that tight control of rat IL-18 expression restricts the production of IFN- in anti-GBM GN in WKY rats, and that IL-18 and IFN- might induce local proliferation of inflammatory cells to amplify renal injury after the treatment with recombinant active IL-18 (rIL-18) of CD8+-mediated M-dependent GN.
METHODS
Construction of cDNA library, screening, and DNA sequencing
Primers for rat IL-18 cDNA were generated from murine cDNA coding for IL-18 (D49949). Polymerase chain reaction (PCR)-cloned rat IL-18 cDNA was used to screen a lipopolysaccharide (LPS)-stimulated rat peritoneal macrophage cDNA library17. To investigate the effect of ICE on the processing of rat IL-18, rat ICE was also cloned from this cDNA library using rat ICE probe prepared from a portion of a high conserved sequence of murine ICE cDNA (U04269).
Anti-GBM Ab-induced glomerulonephritis
Twelve inbred male WKY rats (Charles River Laboratories, Wilmington, MA, USA) weighing 200 to 220 g received one intravenous injection of anti-GBM Ab at a dose of 25 
L/100 body weight; three additional rats were given normal rabbit serum (NRS) and were used as a control (day 0)18. At days 3, 5, 7, and 9, three rats were euthanized at each time point.
Riboprobe generation, RNA extraction, and RNase protection assay
A fragment produced by Xho 1 and Xba 1 (containing the coding region) of IL-18 cDNA was used for preparation of the riboprobe. L-32 (92 bp) was generated by PCR using a cDNA template and was used as a housekeeping gene. Expression of IFN- mRNA was investigated using a cytokine probe set (Pharmingen, San Diego, CA, USA). Glomeruli were isolated by sequential sieving through #60 and #100 wire mesh screens (Small Parts, Inc., Miami, FL, USA) as previously described19,20. The glomeruli collected on the #200 mesh screen contained <10% tubular contamination. After washing, the glomeruli were homogenized in 4-M guanidine isothiocyanate with a sonicator (Heat Systems-Ultrasonics, Plainview, NY, USA). The RNA was prepared by a single-step method21. The RNase protection assay (RPA) was described previously20,22. Phosphoimage quantitation was performed using the PhosphorImager SI scanning instrument and ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA, USA).
Assessment of 5'-UTR IL-18 mRNA expression
Glomerular 5'-UTR IL-18 mRNA expression during anti-GBM GN was determined as described using the Eco R1 and Hind III 5'-UTR fragment of IL-18.
Construction of a reporter gene for luciferase (Luc) assay
A Luc 5UT reporter construct was made to study the role of the IL-18 5'-UTR. Luc 5'-UT, which contains 186 bp of the 5'-UTR of rat IL-18, was constructed as follows: Plasmid pBluscript rat IL-18, which contains the full-length rat IL-18, was digested with Eco RI and Hind III. The Luc-coding region was obtained from the plasmid pGL3 promoter by cutting with Hind III and Xba I, and was subcloned into Hind III and Xba I sites of pBluscript. pCDNA vector was digested with Eco RI and Not I; Luc cDNA was digested with Hind III and Not I. The Eco RI and Hind III 5'-UT fragments were then ligated together to generate pLUC5UT. The Luc-coding region alone was subcloned into Hind III and Xba I sites of pCDNA3 to be used as a control. Electroporated with 10 g of plasmid at 1500 V, 1000 F, and then plated in 3.5 cm plates were 3 
105 cells. The cells were harvested 48 hours later and extracts were prepared for Luc assay as previously described23.
Transfection of rat IL-18 cDNA with or without 5'-UTR in the presence or absence of ICE
COS-7 cells (3 105) were transfected by electroporation with pCDM8IL-18 alone (with or without 5'-UTR) or in combination with an expression plasmid encoding ICE. After 48 hours of incubation, the supernatants were collected and analyzed for the presence of IL-18 protein by Western blot using IL-18 antiserum. The supernatants also were used to assay the biologic activity of IL-18. Total RNA was extracted for RNase protection assay from the cell pellets by the single-step method21.
Expression of rat IL-18 in Escherichia coli and refolding of rIL-18
DNA encoding an active form of rat IL-18 (amino acid 37 to 194) was PCR-amplified and expressed in a His-tagged form following a previously described procedure24,25. IL-18 was refolded on the column using a refolding buffer containing a urea gradient of 4 mol/L to 0.5 mol/L, which was added at a rate of 0.5 mL/min. After refolding, the protein was eluted with 80 mmol/L imidazol/0.5 mol/L urea. A polyclonal antiserum was raised by immunizing a rabbit with rIL-18. After two boosters, the serum was obtained and used for Western blot.
Western blot
The samples were electrophoresed on a 4–12% Nu-Page bis-tris gel (NOVEX, San Diego, CA, USA) and were then transblotted onto a nitrocellulose filter (Schleicher & Schuell, Inc., Keene, NH, USA). The blot was incubated in the rabbit-anti IL-18 Ab solution (1:2000 dilution) and then treated with goat-anti-rabbit immunoglobulin G (IgG) conjugated with alkaline phosphatase (1:1500 dilution; Boehringer Mannheim, Indianapolis, IN, USA)26.
Biologic activity of IL-18
IL-12 (10 pg/mL) primed plastic nonadherent spleen cells (4 106/mL, 0.1 mL/well) were incubated with either recombinant His-tagged rat IL-18 or supernatants derived from transfected COS-7 cells. Following incubation, supernatants were collected and IFN- titers were determined by enzyme-linked immunoassay (ELISA).
Determination of rIL-18 effect during anti-GBM GN
Six additional rats with anti-GBM GN were used to study the effect of active IL-18; three rats received phosphate-buffered saline (PBS), and three were given 50 g of active IL-18 intravenous for 5 days and sacrificed on day 6. As a control, three normal WKY rats received active IL-18 as above. IFN- expression in glomeruli was determined by ELISA. Levels of IFN- were also measured in liver to determine whether the effects of administration of recombinant IL-18 were restricted to nephritic glomeruli. For histopathology study, kidney tissue samples were fixed in 10% neutralized buffered formalin (NBF), or methol-Carnoy fixative solution, and embedded in paraffin. For light microscopy examination, 5 m paraffin sections of NBF-fixed tissue were stained with periodic acid-Schiff (PAS) reagent. The number of crescentic glomeruli per 100 glomeruli of each rat was calculated and expressed as a percentage. Immunohistochemistry double staining of CD8+ and proliferating cell nuclear antigen (PCNA) or ED1+ and PCNA were carried out as previously described16. Proteinuria was assayed by the sulfosalicylic method.
RESULTS
Cloning of rat IL-18 and rat ICE
Rat ICE cDNA corresponded to the published sequence in the GenBank (accession no. U14647). The conceptualized amino acid sequence derived from the rat IL-18 cDNA clone predicts a protein with a molecular mass of 24 kD and an open reading frame that encodes for 194 amino acids as a precursor protein (GenBank accession no. AY077842). The sequence was identical to rat IL-18 published by Culhane et al27. The rat amino acid sequence was 90.2% homologous with mouse IL-18 and 64.9% with human IL-18, respectively. Analogous to these mouse and human proteins, rat IL-18 contains the IL-1 signature sequence. At the putative processing site, a conserved Asp-X sequence in the rat, as well as other species, suggests the importance of this amino acid in the maturation of IL-18 Figure 1.
Figure 1.
Amino acid sequence of rat interleukin-18 (IL-18). Homology of rat IL-18 with mouse and human IL-18 is shown. The IL-1 signature sequence is underlined. The sequence of the putative processing site is indicated by italics (for rat and human Asp36-X and for mouse Asp35-X).
Full figure and legend (81K)A difference among rat IL-18 and mouse and human IL-18 was found in the 5'-UT region; human IL-18 contains 177 bp (GenBank accession no. D49950) and mouse IL-18 contains 164 bp (GenBank accession no. D49949) at the 5'-UT region, respectively. In contrast, the rat IL-18 contains a longer 5'-UT region of 228 bp. At the 5'-UT region the homology between the 164 bp sequence of mouse IL-18 and the 164 bp sequence adjacent to the ATG codon of rat IL-18 was 80.4%, and 33.8% for human IL-18 in 177 bp Figure 2
Figure 2.
Base pair sequence of rat, mouse, and human interleukin-18 (IL-18) 5'-UTR. Arrows represent the fragment used as a riboprobe during RNase protection assay.
Full figure and legend (75K)IFN-
and IL-18 mRNA expression during crescentic GN
A small increase in expression of IFN- mRNA was found in anti-GBM GN in WKY rats, in contrast with high expression of other cytokines such as IL-1
, IL-1, IL-4, IL-6, and TNF-Figure 3a. Ribonuclease protection assay (RPA) using a probe containing the coding region of IL-18, revealed a low basal level of IL-18 mRNA that substantially increased during anti-GBM GN. Maximum expression occurred at days 3 and 5, and then decreased at days 7 and 9 Figure 3b. ELISA analysis of INF- protein confirmed little increase of protein levels of this cytokine in nephritic glomeruli (control vs. anti-GBM GN, 100 
20 vs. 180.66 35.2 pg/mL, P> 0.05) and correlated with the low expression levels of IFN- mRNA during this model of GN.
Figure 3.
Analysis of interferon gamma (IFN-) and interleukin-18 (IL-18) expression in the glomeruli of crescentic glomerulonephritis (CGN) in Wistar Kyoto (WKY) rats. (A) RNase protection assay of cytokine set. Eleven cytokines were tested, and L-32 and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were used as housekeeping genes. Only cytokines expressed are shown. (B) RNase protection analysis of IL-18 coding region. IL-18 mRNA expression is expressed as the ratio of IL-18 to L32 X 10. (C) RNase protection assay of IL-18 5'-UTR region. The figure shows two different transcripts, one of 180 bp and the second 50 bp shorter. Probes contain polylinker regions and are longer than the protected bands. Each lane represents one rat sample.
Expression of 5'-UTR IL-18 mRNA in anti-GBM GN
When a probe with the 5'-UTR of rat IL-18 was tested, we found that most of the mRNA for IL-18 was the 5'-UT containing transcript Figure 3c. Two different transcripts of the 5'-UTR were expressed during anti-GBM GN, one transcript of 180 bp that was not constitutively expressed, and a second transcript that was 50 bp shorter. Peak expression occurred at days 3 and 5 for both transcripts Figure 3c.
The role of 5'-UTR in the translational control of IL-18
To address the role of the 5'-UTR of rat IL-18 mRNA in modulating the translation of this message and to explain the dissociation between high expression of IL-18 gene and low expression of IFN- mRNA during anti-GBM GN, several experiments were performed. In the first experiment, the expression of IL-18 with or without 5'-UTR in COS-7 cells was determined. For this purpose, a specific anti-IL-18 Ab was generated for Western blot. This Ab recognized IL-18 expressed in COS-7, but did not recognize any protein in supernatants from antisense transfectants Figure 4a. We found that only coexpression of pro-IL-18 and ICE without the 5'-UTR resulted in the cleavage of the 24 kD pro-IL-18 into the expected 18 kD mature IL-18 fragment. In the presence of 5'-UTR, neither 18 kD nor 24kD pro-IL-18 could be detected by Western blot Figure 4a. The RPA showed that IL-18 mRNA was transcribed with no difference in COS-7 cells transfected with IL-18, whether the 5'-UTR region was present or not Figure 4b.
Figure 4.
(A) Protein expression of interleukin-18 (IL-18) in transfected COS-7 cells. Cleaved pro-IL-18 is demonstrated in the supernatant from COS-7 cotransfected with IL-18 and ICE (IL-18 + ICE) without the 5'-UTR. The upper band is the remainder of undigested pro-IL-18. IL-18 protein expression was found in the supernatant of IL-18 without 5'-UTR transfectant in two different samples [5'-UTR(–) a and b], whereas no protein expression was found in supernatant from COS-7 cells transfected with IL-18 in the presence of 5'-UTR [5'-UTR(+)]. Recombinant rat IL-18 expressed in E. coli and refolded, showing a larger size than the expected 18 kD due to the extra AA fused with IL-18 during expression. (B) RNase protection assay of COS-7 transfected with IL-18 in the presence or absence of IL-18 5'-UTR.
Full figure and legend (108K)These data suggested the presence of an IL-18 5'-UTR translational inhibitor. To demonstrate that this translational effect was specific to the IL-18 leader sequence, we made a chimeric gene in which IL-18 5'-UTR was fused with the luciferase reporter gene; the results of duplicate assays demonstrated that the construct containing the rat IL-18 5'-UTR inhibited Luc gene expression by 35.4% in a chimera [706,381 integrated light units (ILU) against l,093,545 ILU for the plasmid without the IL-18 5'-UTR]. Furthermore, when the bioactivity of IL-18 was determined, supernatants from COS-7 coexpressing ICE and pro-IL-18 without 5'-UTR could stimulate IL-12–primed nonadherent splenic cells to produce IFN-; conversely, cells cotransfected with ICE and pro-IL-18 containing 5'-UTR only produced minimal levels of IFN-Figure 5.
Figure 5.
Biologic activity of interleukin-18 (IL-18) of supernatants derived from transfected COS-7 cells with IL-18 (with or without 5'-UTR) and interleukin-1
converting enzyme (ICE). Recombinant rat IL-18 at different concentrations and supernatant from the macrophage cell line Raw 264.7 (at dilutions of 1:40 and 1:400) were used as positive control. Minimal production of interferon gamma (IFN-) was found in the supernatants from COS-7 cells coexpressing ICE and pro-IL-18 in the presence of 5'-UTR [ICE/5'UTR (+)] in contrast with the high bioactivity of transfectant without 5'-UTR [ICE/5'UTR (-)], using the same dilutions (1:4 and 1:40). A little background was found in the transfectant of ICE alone.
Determination of IL-18 protein expression in nephritic glomeruli by Western blot resulted in the lack of detection of IL-18 protein, supporting the inefficient translation of IL-18 mRNA by its 5'-UTR. These results suggest that the 5'-UTR of rat IL-18 is a potent inhibitor of translation.
Effect of recombinant IL-18 in rats with anti-GBM GN
To investigate the potential role of IL-18 during GN, a biologically active rat IL-18 was injected for 5 days into WKY rats with anti-GBM GN. Rats treated with rIL-18 showed a 2.4-fold higher IFN- production in glomeruli (443.33 39.5 vs. 180.66 35.2 pg/mL, P < 0.05). This led to a 3.1-fold increase of proteinuria (37.2 0.72 vs. 12.3 0.58 mg/24 hours, P < 0.001). The frequency of crescentic glomeruli was dramatically increased in rats treated with rIL-18 (85% vs. 55%, P < 0.001) Figure 6.
Figure 6.
(a,c,e) Photomicrograph of glomeruli from Wistar Kyoto (WKY) rats with crescentic glomerulonephritis (CGN) that were treated with phosphate-buffered saline (PBS) or (b,d,f)rIL-18. (a to d) Kidney section immunohistochemistry stained for ED1+/Mo/M (magnification of a and b, 200; magnification of c and d, 400). (e) Periodic acid-Schiff (PAS) staining of kidney section of PBS- or (f) rIL-18-treated rats.
Double immunohistochemistry staining of ED1+M or CD8+ cells and the PCNA showed a large number of proliferating M (ED1+PCNA+) (29.8 5.6 vs. 17.8 3.7 per glomerular cross-section, P < 0.01) and CD8+ cells (CD8+PCNA+) (7.6 1.2 vs. 5.8 1.8 per glomerular cross-section, P < 0.05) Figure 7. Proliferating cells were localized in areas of severe tissue damage. These data demonstrated that when the suppression of translation of IL-18 is bypassing using rIL-18, an increase of IFN- is induced in this model of GN, with worsening renal damage.
Figure 7.
Photomicrograph of glomeruli from Wistar Kyoto (WKY) rats with crescentic glomerulonephritis (CGN) treated withrIL-18. (a and b) Kidney sections were immunohistochemically stained for CD8+ and proliferating cell nuclear antigen (PCNA) and (c and d) macrophages (M) and PCNA. (b) and (d) Magnification of marked areas from (a) and (c), 400. Blue arrows indicate PCNA staining; brown arrows indicate staining of inflammatory cells; and black arrows denote costaining of PCNA and inflammatory cells.
In contrast to the increased levels of INF- in the nephritic glomeruli after the treatment with rIL-18, levels of liver IFN- were not detectable in rats that received rIL-18 or PBS.
In healthy rats, treatment with IL-18 caused no damage in the kidney; consequently, urine protein levels were not different from those observed in normal WKY that did not receive IL-18 (3.3 vs. 2.7 mg/24 hours).
DISCUSSION
IFN-, a cytokine secreted by activated T cells and NK cells, has immunomodulatory effects on several cell types28. IFN- is a cytokine that activates M; in addition, IFN- contributes to the regulation of the T-helper cell immune response12,28. NK cells exhibit enhanced cytolitic activity in response to IFN- in vivo and in vitro12,28. IFN- cytokine plays a role in several autoimmune diseases, including GN. It has been demonstrated that levels of IFN- in the sera of patients with autoimmune disorders are significantly elevated29. Inhibition of IFN- or treatment with soluble IFN-R attenuates F1 lupus–like nephritis29,30,31. In addition, in a model of anti-GBM GN in Sprague-Dawley rats and in C57BL/6 mice, significantly increased levels of IFN- were detected32. In contrast with other models of GN, low levels of IFN- mRNA and protein expression were found during anti-GBM GN in WKY rats, despite the presence of IL-18 gene expression, a potent inducer of IFN- production. We found the presence of a long 5'-UTR in rat IL-18; when IL-18 was transfected in the presence of its 5' flanking sequence, IL-18 protein could not be detected. Furthermore, the construction of an expression vector in which IL-18 5'-UTR was placed flanking the luciferase coding sequence, the luciferase gene expression was inhibited, suggesting the presence of an IL-18 5'-UTR translational inhibitor. This translational inhibitor was highly expressed in anti-GBM GN in WKY rats and correlated with a low expression of IFN- mRNA and lack of detection of IL-18 protein levels in nephritic glomeruli. These data suggest that IFN- and IL-18 play a minimal role in this model of GN. Conversely, the treatment with rIL-18 of WKY with anti-GBM GN induced an increase of glomerular IFN- associated with a more severe glomerular damage, although no effect was observed in control WKY rats. IL-18 administration in mice without GN did not alter glomerular histology has also been previously reported33. The lack of effect of IL-18 in healthy WKY rats is consistent with previous findings, in which in mice not primed, the administration of IL-18 does not increase plasma levels of IFN- or induce damage in the liver or other organs. However, induction of obstructive jaundice prior to the treatment with IL-18 induces large amounts of IFN- and liver injury, and the combined treatment of IL-18 plus IL-12 in mice not primed induces elevated levels of IFN- and liver damage4,34,35. These data suggest that a priming phase that activates the immune system should precede a late excitation phase elicited by IL-18 in order to induce IFN- production, leading to tissue injury. It is also suggested that IL-18 is a cofactor in the induction of IFN- production, since treatment with IL-18 and IL-12, but not with IL-18 alone, stimulates the production of INF-. However, whether longer time treatment with IL-18 alone without priming phase could cause tissue injury remains to be determined. In spite of the deleterious effects of the treatment with IL-18 in the kidney of anti-GBM rats, IFN- levels in the liver were not affected in these rats. This preferential effect of IL-18 in nephritic glomeruli supports the theory that a preceding inflammatory insult is necessary for IL-18 to augment IFN- production with a further tissue injury4,34,35.
The presence of the 5'-UTR and/or 3'-UTR regions affects translational efficiency and in turn can be a mechanism of gene regulation36. Two factors that have been demonstrated to influence the translatability of mRNA are present in rat IL-18: extensive structure in the 5'-UTR and the presence of a minicistron, a short open reading frame (ORF) upstream of the actual translation start site36. It is probable that the long 5'-UTR forms a secondary structure, such as a hairpin, which in turn inhibits translation in a position-dependent mechanism. There are other examples of a 5'-untranslated leader sequence acting as a potent translational inhibitor. In bovine aldehyde dehydrogenase, platelet-derived growth factor-2, and ornitine decarboxylase, the 5'-UTR suppresses translation of its own coding sequence. In all three cases, very stable hairpin structures have been predicted by computer analysis37,38,39. However, other potential mechanisms besides the presence of stable steam loops for repressing translation should be considered. The formation of mRNA-protein complexes in the 5'-UTR has been described to control translation initiation by steric blockage of a sensitive step in the initiation pathway. As an example, HuR, a related member of the human Hu/Elav protein family (a family of RNA-binding factors) that is ubiquitously expressed, was previously identified as a protein binding to a U-rich element in the p27 5'-UTR to inhibit its translation40,41. It will be interesting to investigate whether IL-18 mRNA contains HuR binding sites in its 5'-UTR.
The presence of two different lengths of IL-18 5'-UTR in this model of GN suggests that they arise from alternative splicing. It is probable that the shorter transcript may correspond to a nonfunctional, silent transcript by retaining at least part of one intron.
This study indicates that rat IL-18 is regulated at multiple steps, including transcriptional, post-transcriptional, translational, and post-translational levels. Recently it has been demonstrated that pro-IL-18 and mature IL-18 are degraded to biologically inactive forms by caspase-3 in monocytic THP.1 cells. Because of the detrimental effects in the case of overexpression or lack of expression of IFN- and IL-18, it would be advantageous to have IL-18 under strict regulatory control. Similar tight control is observed with another cytokine, TNF-, in which diverse cis-acting elements and transacting factors are involved in TNF- gene expression. A cell type–specific mechanism for control of this potent cytokine has also been suggested42. Recently it has been demonstrated that the adrenal gland, spleen, and duodenum express different lengths of the 5'-UTR, indicating tissue-specific usage of the promoter region of rat IL-1843. However, whether a cell-type gene regulation is involved in IL-18 expression remains to be further determined.
The enhanced glomerular injury after treatment with rIL-18 in WKY rats with anti-GBM GN correlated with an increase in IFN- levels and CD8+ and ED1+ cell proliferation. Proliferating cells PCNA ED1+ and PCNA CD8+ were localized in areas of severe tissue damage. Anti-GBM GN in WKY rats is characterized by CD8+ lymphocyte–dependent and M-related glomerular injury; accordingly, the proliferation of these cells might contribute in part in the severity of the glomerular lesions in rats treated with rIL-18. There is evidence to support that local proliferation is a mechanism for amplifying renal injury, since the degree of proliferating M and interstitial proliferating T cells correlates with the severity of histologic damage and with impairment of renal function in different models of kidney injury44,45. Previously, it has been shown that IFN- promotes M proliferation in mesangial proliferative GN46, in addition to recruiting CD8+ T cells and M. IL-18 enhances the production of GM-CSF, which has the ability to induce local M proliferation. IL-18 is able to induce the proliferation of activated T cells, and could be responsible for the proliferation of CD8+ cells. Thus, both IFN- and IL-18 could mediate the local proliferation of ED1+ and CD8+ cells observed after the treatment of WKY rats with anti-GBM GN with rIL-18; however, further studies are needed. There are additional factors by which IL-18 and IFN- might influence the severity of this nephritis after treatment with rIL-18. IL-18 and IFN- exhibit proinflammatory properties by inducing the expression of cytokines, adhesion molecules, and chemokines47. All of these mediators play a critical role in the pathogenesis of anti-GBM GN in WKY rats18,48. In addition, IL-18 directly up-regulates perforin-mediated NK activity and activates CD8+ cytotoxic lymphocytes. However, whether the glomerular damage is due to secondary mediators localizing to the kidney or by an IL-18/IL-18 receptor interaction remains to be determined.
CONCLUSION
These results indicate that tight translational control of rat IL-18 expression restricts the production of IFN- in anti-GBM GN in WKY rats, and that IL-18 and IFN- might induce local proliferation and infiltration of inflammatory cells in order to amplify renal injury after the treatment with rIL-18 of CD8+-mediated M-dependent GN.
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Acknowledgments
This is publication No. 14382-IMM from the Department of Immunology, The Scripps Research Institute, La Jolla, California. This work was supported in part by NIH Grant Nos. DK54674-02 (L.F.) and DK-20043 (C.B.W.). Dr. Xia was the recipient of a fellowship from The National Kidney Foundation of Southern California.
