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J Am Soc Nephrol 14:2823-2832, 2003
© 2003 American Society of Nephrology

BASIC SCIENCE

Differential Usage of Class II Transactivator Promoters PI and PIV during Inflammation and Injury in Kidney

Oki Takeuchi*, Tasha N. Sims{dagger}, Yutaka Takei{dagger}, Vido Ramassar{dagger}, Konrad S. Famulski{dagger} and Philip F. Halloran{dagger}

*Department of Medical Microbiology and Immunology, and {dagger}Department of Medicine, Division of Nephrology and Immunology, University of Alberta, Edmonton, Alberta, Canada.

Correspondence to Dr. Philip F. Halloran, Division of Nephrology & Immunology, University of Alberta, 250-Heritage Medical Research Centre, Edmonton, Alberta T6G 2S2, Canada. Phone: 780-407-8880; Fax: 780-407-3417;


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Expression of class II transactivator (CIITA), the transcriptional regulator that controls all class II expression, is controlled in cell lines in vitro by three promoters: the dendritic cell promoter PI, the B cell promoter PIII, and the interferon-{gamma} (IFN-{gamma})–inducible promoter, PIV. The authors examined the promoter usage in vivo in mouse kidney in the basal state and in response to IFN-{gamma}, endotoxin, allostimulation, and renal injury. Genetically modified mice were used to examine the dependency of each promoter on IFN-{gamma} and on the transcription factor interferon regulatory factor 1 (IRF-1). Usage of distinct CIITA promoters was monitored by real-time reverse transcriptase polymerase chain reaction (RT-PCR) using the unique sequences in the 5' end of the transcript from each promoter. Kidneys in both control mice and IFN-{gamma} knockouts expressed chiefly PI- and PIV–related products. Administration of recombinant IFN-{gamma} activated only promoter PIV. Endotoxin or allogeneic stimulation elevated the PIV-related mRNA, dependent on IFN-{gamma} and on IRF-1. Ischemic renal injury, however, increased the PI- and PIV–driven mRNA expression in wild-type but also in IFN-{gamma}–deficient mice. Thus the in vivo control of CIITA promoters in kidney is similar to that observed in vitro (i.e., basal-state usage of PI and IFN-{gamma}–dependent usage of PIV during inflammation), but it also shows additional levels of control: IFN-{gamma}–independent basal activity of PIV and IFN-{gamma}–independent induction of PIV during tissue injury. E-mail: phil.halloran@ualberta.ca


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MHC class II molecules are important in CD4 T cell development and in antigen presentation during the immune response in infection, autoimmunity, and graft rejection. Class II proteins are expressed on antigen-presenting cells such as B cells, dendritic cells, and macrophages. Class II expression in vivo in peripheral tissues may play a role in T cell homeostasis, maintaining the peripheral CD4 T cell population (1,2). In addition, in human kidney in the basal state, a variable number of epithelial and endothelial cells express class II (3), perhaps due to basal production of interferon-{gamma} (IFN-{gamma}) (4). Expression of class II in mouse kidney endothelium and epithelium can be induced de novo by IFN-{gamma} (5,6).

The master regulator of cell-specific and IFN-{gamma}–inducible class II expression is the class II transactivator (CIITA) (7–10). CIITA belongs to the NACHT protein family, the members of which are involved in inflammation and apoptosis (11). CIITA mediates its function through specific interaction with DNA-bound proteins rather than by direct binding to DNA (12). The cloning of the human and mouse CIITA 5' regulatory region revealed three nonhomologous promoters: PI, PIII, and PIV (13–15). These promoters direct the synthesis of three distinct transcripts with unique 5' regions. Promoter I (PI) is activated in dendritic cells and directs the constitutive expression of the 132-kD isoform. Promoter III (PIII) product, a 124-kD isoform, is mainly detectable in B cell lines (14,16) and is silenced with maturation to plasma cells (17). Promoter IV (PIV) is the IFN-{gamma}–inducible promoter in vitro (14,18), and it has multiple transcription factor binding sites, including STAT1, E-box, and interferon regulatory factor 1 (IRF-1) sites. It produces the 121-kD isoform that is crucial for the expression of MHC class II in nonhematopoietic cells (19).

Functions of the CIITA promoters have been studied primarily in vitro, leaving the in vivo role of each promoter in inflammation or tissue injury unknown. Given the potentially important role of class II in renal disease and transplantation, we examined in the present studies the control of class II expression in kidney by measuring the relative activity of each promoter in the basal and stimulated states using real-time reverse transcriptase polymerase chain reaction (RT-PCR). The results confirm that CIITA regulation in kidney in vivo follows the principal rules identified in cell cultures, but they also reveal additional levels of control.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Mice with disrupted IFN-{gamma} gene (GKO) were created in the BALB/c background (20). Heterozygous GKO mice were generously provided by Dr. T. Stewart (Genentech Inc., South San Francisco, CA). Mice with disrupted IRF-1 gene (IRF-1 KO) were generated by disrupting the IRF-1 gene in the 129/J background (21). Homozygous IRF-1 knockout (KO) mice were a gift from Dr. T. W. Mak (Ontario Cancer Institute, Toronto, ON, Canada). Wild-type (WT) female mice, 6 to 10 wk old (WT) BALB/c (BALB/cCr/AltBM) were obtained from the Health Science Laboratory Animal Services (HSLAS) at the University of Alberta and were used as matched controls for GKO mice. 129/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were used as matched controls for IRF-1 KO mice. Mice were kept on acidified water to inhibit the growth of bacteria in the water source, a standard practice in mouse colony. Animal maintenance and experiments were in conformity with approved animal care protocols.

IFN-{gamma} Treatment
Recombinant IFN-{gamma} (rIFN-{gamma}) was generously provided by Dr. T. Stewart (Genentech Inc., South San Francisco, CA). Five mice per each time point received 100,000 IU of rIFN-{gamma} in a single intraperitoneal injection and were killed at 2, 4, 12, 24, 48, 72, or 96 h after injection. Kidneys were then immediately removed, decapsulated, snap-frozen in liquid nitrogen, and kept at -70°C.

P815 Tumor Allografts
Murine mastocytoma tumor cell line P815 (ATCC, American Type Culture Collection, Rockville, MD) was passaged in DBA/2 (H-2d) mice (Jackson Laboratory). After P815 cells were collected from the ascites fluid, 20 x 106 cells were injected intraperitoneally into five mice per experimental group. Kidneys were harvested on day 7.

Endotoxin Treatment
Endotoxin (LPS) from Salmonella minnesota (Sigma, St. Louis, MO) was dissolved in sterile saline at 250 µg/ml and heated at 56°C for 5 h. Five mice per experimental group were injected intraperitoneally with either saline (control) or LPS (25 µg), and kidneys were harvested at 1, 3, 6, 12, 24, 48, 72, or 96 h after injection.

Ischemic Acute Tubular Necrosis
Ischemic injury to the kidney was achieved by clamping the left renal pedicle for 60 min. Sham-operated control mice underwent a simple laparotomy under identical conditions.

Radioactive Antibody-Binding Assay
This technique has been previously reported, and its quantitative characteristics have been described (22). Briefly, kidney transplant specimens were homogenized, washed in phosphate-buffered saline (PBS), and centrifuged at 3000 rpm for 20 min. The pellets were suspended in PBS at 20 mg/ml. Five milligrams of extract was then centrifuged and resuspended in 100 µl of radiolabeled mAb solution (1000 cpm/µl) and incubated on ice for 60 min. After that time, incubates were diluted with 1 ml of PBS and centrifuged at 3000 rpm for 20 min. The pellets were counted in a gamma counter and the values were corrected for the nonspecific binding.

Analysis of mRNA by Real-Time Quantitative RT-PCR
Total RNA was prepared from tissues as described previously. Two micrograms of RNA was transcribed using M-MLV reverse transcriptase and random primers. Quantification of gene expression was performed using the ABI prism 7700 Sequence Detection System (PE Applied Biosystems) as described previously (23). All TaqMan probe/primer combinations were designed using Primer Express software version 1.5 (PE Applied Biosystems) and are listed in Figure 1. Hypoxanthine phosphoribosyltransferase (mHPRT) primers and probe sequences were as follows: sense 5'-TGACACTGGTAAAACAATGCAAACT-3', antisense 5'-AACAAAGTCTGGCCTGTATCCAA-3', probe 5'-TTCACCAGCAAGCTTGCAACCTTAACC-3'.



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  		Figure 1. Primers and probes designed for quantitative reverse transcriptase polymerase chain reaction (RT-PCR) of class II transactivator (CIITA). (Upper panel) positions of the forward primers within the three different 5' ends of the CIITA mRNAs and position of the common probe/common reverse primer in the second exon are shown. Coding regions are represented by wide boxes, and 5'-untranslated regions by narrow boxes. The non-homologous regions are hatched differently. Open bars represent the size (bp) of each amplicon and also the synthesized oligo-nucleotides used for the amplification efficiency control. Relevant sequences are given below the scheme. (Lower panel) amplification efficiency control for primer/probe sets designed to detect PI, PIII, and PIV products.

 
Probes were labeled at the 5'-end with the reporter dye molecule FAM (6-carboxy-fluorescein) or VIC, respectively, and at the 3'-end with the quencher dye molecule TAMARA (6-carboxy-tetramethyl-rhodamine). Each 25-µl reaction consisted of 1 x TaqMan Universal PCR Master Mix (PE Applied Biosystems), 300 nM forward primer, 300 nM reverse primer, 200 nM TaqMan quantification probe, and 25 ng of cDNA template. PCR amplification conditions were as follows: 50°C for 2 min, 95°C for 10 min, then 40 cycles of 95°C for 15 s and 62°C for 1 min. Emitted fluorescence for each reaction well was measured every cycle during both the denaturation and annealing/extension phases. Amplification plots were constructed using ABI PRISM 7700 Sequence Detection System software version 1.7 (PE Applied Biosystems). Threshold cycle numbers (Ct) were determined and transformed using the {Delta}Ct or {Delta}{Delta}Ct methods as described by the manufacturer, using hypoxanthine phosphoribosyltransferase (mHPRT) as the calibrator. All PCR assays were performed in duplicates and reported as the average. To estimate the total CIITA expression, we used a mixture of all three forward primers, the common reverse primer, and the common probe. All data were expressed as relative to the total CIITA expression in a control or sham kidney.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Primers and Probes Used in Quantitative RT-PCR
To prevent amplification of the genomic DNA, we selected specific forward primer sequences for transcripts from each class II transactivator (CIITA) promoter that spanned exon-intron boundaries between the first and second exon. Sequences for the common reverse primer and the common probe were selected from the second exon of CIITA that is shared by all isoforms (Figure 1A). We confirmed that the amplification efficiency of the three primer/probe sets was similar by synthesizing three oligonucleotides corresponding to sequences determined by our primer sets and using them as templates in the assay (Figure 1B).

Basal Usage of CIITA Promoters in Mouse Tissues
We previously reported that CIITA mRNA expression in vivo correlates with the distribution of class II molecules in mouse nonlymphoid organs (24). To understand the role of each promoter in basal expression, we evaluated the activities of promoters PI, PIII, and PIV in tissues from untreated mice, using quantitative real-time RT-PCR (Figure 2). Relative expression of mHPRT, our reference gene, was very similar in all tissues. Total CIITA mRNA was highly expressed in the spleen, approximately 65 times more than in the kidney. Promoter PI was the major contributor to total CIITA mRNA expression in skin, compatible with the known restriction of expression in skin to the Langerhans dendritic cells (14). PIII, which governs expression in B cells, was highly active in the spleen and poorly used in all nonlymphoid tissue. PIV was modestly active in spleen and kidney. Activity of PI and PIV in kidney was similar and about 14 times higher than PIII (Figure 3). PI activity in the basal state in kidney was expected due to interstitial cells of dendritic lineage (25). Unexpectedly, the IFN- {gamma}–inducible promoter PIV was active in kidneys from the untreated mice, suggesting either a response to steady-state level of IFN-{gamma} production or non-IFN-{gamma} induction of PIV.



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  		Figure 2. Relative activity of CIITA promoters in mouse tissues. RNA was extracted from kidney, spleen, liver, heart, brain, and skin of five 129/J mice in the basal state. The RNA was pooled and analyzed for CIITA expression by TaqMan real-time PCR. Shown are ratios relative to the total CIITA expression in kidney. Experimental error was less than 10%. Kid, kidney; Spl, spleen.

 


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  		Figure 3. Basal activity of CIITA promoters in wild-type (WT), interferon-{gamma} (IFN-{gamma})–deficient (GKO), and interferon regulatory factor 1 (IRF-1)–deficient (IRF-1KO) mouse kidneys. Ten mice were analyzed in each group. Data are shown as means ± SD. Expression of CIITA in GKO mice was relative to BALB/c mice, whereas IRF-1KO mice were compared with 129/J mice.

 
We therefore investigated the role of IFN-{gamma} in CIITA promoter induction in WT and GKO mice in the basal state. As shown in Figure 3, mRNA levels for PI- and PIV–driven products were similar in GKO and in WT mice, indicating that the steady-state activity of PIV was IFN-{gamma}–independent. On the other hand, the IRF-1 KO mice presented significantly reduced usage of PI and PIV compared with WT mice.

Effect of rIFN-{gamma} Administration on CIITA Promoter Expression in Kidney
IFN-{gamma} induces the expression of CIITA mRNA and class II mRNA in vivo (6,24) and in vitro (14). To test whether IFN-{gamma} regulates PIV in vivo, we administered rIFN-{gamma} and harvested the kidneys at 2 h to 96 h after injection (Figure 4A). The rIFN-{gamma} induced a peak of class II protein expression at 48 h, as indicated by the radiolabeled antibody binding assay (1698 ± 199 cpm for control kidney extracts versus 2892 ± 492 cpm for treated animals; P = 0.002; n = 5). Total CIITA mRNA expression, however, increased approximately 30-fold at 4 h and returned to the basal level by 24 h. PIV-driven mRNA expression showed a similar pattern. Promoters PI and PIII did not contribute to mRNA expression after IFN-{gamma} administration.



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  		Figure 4. Effect of rIFN-{gamma} on PIV activity in kidney. (A) Time course of recombinant IFN-{gamma} (rIFN-{gamma})–dependent expression of CIITA. BALB/c mice were injected with 100,000 U of rIFN-{gamma}, and their kidneys were harvested at indicated times. Sh, sham kidney. (B) Dependence of CIITA expression on IRF-1. 129/J and IRF-1 KO mice were injected with 100,000 U of rIFN-{gamma}, and the kidneys were harvested at 4 h. Expression of CIITA in IRF-KO mice was relative to 129/J mice. Five mice per each group were used, and the extracted RNA was pooled. Assays were done in duplicates. Experimental error was less than 10%.

 
Role of IRF-1 in Basal and IFN-{gamma}–Induced Usage of CIITA Promoters
Transcription factor IRF-1 is necessary for optimal induction of CIITA mRNA and class II expression by rIFN-{gamma} (26). To address the effect of IRF-1 on the activation of each CIITA promoter in vivo, we injected WT and IRF-1 KO mice with rIFN-{gamma} and harvested kidneys 4 h later, at the peak of CIITA mRNA expression. As expected, rIFN-{gamma} induced PIV-dependent and total CIITA mRNA only in the kidneys of WT mice (Figure 4B).

Class II induction in tubular epithelial cells is less in IRF-1 KO kidneys (26). Figure 4B shows that rIFN-{gamma} induced little PIV in kidneys of IRF-1KO mice. Thus the contribution of IRF-1 to CIITA mRNA induction is attributable to its interaction with promoter PIV.

Effect of LPS on CIITA mRNA Expression
LPS strongly induces class II mRNA and protein expression in kidney epithelium (6,24). To explore the role of CIITA promoters in this response, we administered LPS to BALB/c mice and examined their kidneys at various times (Figure 5A). LPS predominantly affected PIV, with distinct peaks of PIV induction and total CIITA mRNA expression at 12 and 96 h. A small increase in PI mRNA usage occurred at 96 h, rising approximately three-fold to five-fold above basal levels. PIII was not affected by LPS administration at any time.



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  		Figure 5. LPS-induced CIITA mRNA expression in WT and IFN-{gamma}–deficient (GKO) kidney. (A) BALB/c mice were injected with LPS (25 µg), and their kidneys were harvested at the times shown. (B) BALB/c and GKO mice were injected with LPS, and the kidneys were harvested at 12 h and 96 h. Five mice per each group were, used and the extracted RNA was pooled. Assays were done in duplicates. Experimental error was less than 10%. Sh, sham kidney.

 
We examined whether the increase in CIITA mRNA and PIV usage caused by LPS was IFN-{gamma}–dependent. WT and GKO mice were treated with LPS, and kidneys were analyzed at 12 and 96 h (Figure 5B). PIV-driven CIITA mRNA and total CIITA mRNA were increased approximately 13-fold in WT mice in response to LPS injection at 12 h and 8-fold to 10-fold at 96 h. Kidneys of GKO mice showed little change in PIV or total CIITA expression at either time. GKO mice also failed to show the small increase in promoter PI mRNA at 96 h. Thus the major effect of LPS on CIITA and class II expression is probably due to IFN-{gamma}–dependent induction of PIV.

Induction of CIITA Promoters by Allogeneic Stimulation
Allostimulation strongly induces class II expression in kidney via the systemic effects of IFN-{gamma} and other cytokines (27). Unfortunately, it is difficult to study CIITA expression in kidney during kidney rejection because of the extreme diversity of cell types in the infiltrate. However, we previously showed that rejection of the allogeneic tumor cells (P815) in the peritoneal cavity induces a massive class II expression in kidney tubular epithelium and vascular endothelium by day 7, dependent on IFN-{gamma} and IRF-1 (6,26,28). We therefore examined the systemic effect of allostimulation on CIITA promoters. WT, GKO, and IRF-1 KO mice were injected with P815 cells on day 0, and the kidneys were harvested on day 7. In the WT kidney allogeneic stimulation induced total CIITA mRNA expression and activated PIV 18-fold and 15-fold, respectively (Figure 6A). PI-dependent mRNA synthesis was smaller (approximately 5-fold) (Figure 6A). There was no induction of total CIITA mRNA or PI-, PIII-, and PIV–related mRNA in the kidneys harvested from GKO mice, consistent with the previous observations (6,24). IRF-1 KO mice, however, showed the induction of total CIITA-, PI-, and PIV–dependent mRNA. The extent of induction was at least 50% of that observed in WT mice (Figure 6B). Thus CIITA induction during allostimulation correlates mainly with PIV induction but with a small component of PI induction.



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  		Figure 6. Induction of CIITA promoter IV in the kidney after allogeneic stimulation. (A) CIITA expression in IFN-{gamma}–deficient (GKO) and (B) IFR-1–deficient (IRF-KO) mice. BALB/c, GKO, 129/J, and IRF-1 KO mice were injected with 20 x 106 P815 cells on day 0, and their kidneys were harvested on day 7. Expression of CIITA in GKO mice was relative to Balb/c mice (A), and IRF-KO mice were compared with 129/J mice (B). Five mice per each group were used, and the extracted RNA was pooled. Assays were done in duplicates. Experimental error was less than 10%. Sh, sham kidney.

 
IFN-{gamma} Independent Activation of CIITA Promoters in the Kidney by Ischemic Injury
Renal injury induces expression of class II and CIITA, involving both IFN-{gamma}–dependent and IFN-{gamma}–independent mechanisms (6,29). We examined the effect of renal injury on individual CIITA promoters. Left kidney was cross-clamped for 60 min to induce ischemic acute tubular necrosis (ATN), while the untreated right kidney served as a control. When the kidneys were examined after 7 d, the injured left kidney showed approximately 3-fold increase in total CIITA mRNA in both WT and GKO mice (Figure 7), consistent with the previously reported increases in class II products (3,6). ATN induced not only PIV-dependent mRNA but also PI-dependent mRNA. Even in GKO mice, the kidney with ATN displayed increased levels of PI and PIV mRNA (Figure 7). Thus in ischemic injury the induction of CIITA mRNA was due to the increase of PI and PIV usage independent of IFN-{gamma}.



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  		Figure 7. Activation of CIITA promoters after ischemic injury. Ischemic injury to the left (L) kidney was inflicted for 60 min and was compared with the unclamped right (R) kidney. Control mice underwent a simple laparotomy under identical conditions. The kidneys were harvested on day 7. Five mice per each group were used, and the extracted RNA was pooled. Promoter activity is given as a ratio relative to a sham right kidney in each group. Assays were done in duplicates. Experimental error was less than 10%. ATN, acute tubular necrosis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Class II induction in kidney accompanies many disease states and is one of the first examples of regulated gene expression in kidney. In this study, we explored the expression of individual CIITA promoters in kidney in the basal state and in response to stimuli known to induce class II. The behavior of the CIITA promoters in vivo was consistent with their roles previously suggested by studies in cell lines, but it also pointed to additional levels of control. The relative levels of promoter-specific mRNAs varied in different mouse tissues in the basal state. Spleen expressed significant levels of mRNA from PI and PIII promoters, consistent with its content of dendritic cells and B cells, whereas skin displayed only PI mRNA, consistent with its high content of Langerhans cells of dendritic lineage. Similarly, kidney expressed promoters PI as expected from its content of class II–positive interstitial cells of dendritic lineage. On the other hand, both spleen and kidney showed expression of the IFN-{gamma}–inducible PIV mRNA. At least in kidney this was independent of IFN-{gamma}, indicating that factors other than IFN-{gamma} can induce PIV. Treatment with rIFN-{gamma} or stimuli that trigger endogenous IFN-{gamma} production induced high levels of PIV mRNA in kidney. B cell–specific promoter PIII, on the other hand, showed negligible basal or induced mRNA in kidney. There were, however, novel forms of control in vivo not previously described: IFN-{gamma}–independent basal expression of PI and PIV mRNA in kidney and IFN-{gamma}–independent activation of PIV after ischemic injury.

Basal expression of CIITA mRNA in kidney was IFN-{gamma}–independent and was driven by promoters PI and, surprisingly, PIV. Immunostaining of mouse kidney in the basal state shows that class II is confined to the interstitial cells, and is largely IFN-{gamma}- and IRF-1–independent (6,30). Thus PI and PIV are probably activated in the interstitial cells. Renal interstitial cells are related to dendritic cells, but most lack maturation markers such as DEC 205, -881/87 and -87–2 negative (data not shown) (31,32). Moreover, mature dendritic cells are rare in normal kidney in the basal or induced states (33,34). Other species may differ from mice in their use of the CIITA promoters in nonlymphoid organs; for example, some humans and some rat strains express class II in epithelial and endothelial cells (35).

It was surprising to find that the expression of class II in kidney in the basal state was independent of IFN-{gamma}, given that expression of class I molecules in mouse and human nonlymphoid tissues is partially dependent on the steady-state production of IFN-{gamma} (3,4,6). Tissues such as kidney are exposed locally to low levels of IFN-{gamma} in the basal state, but class II is not induced. This is probably a dose issue; class I is more readily induced by IFN-{gamma} than is CIITA and therefore class II. Nevertheless, PIV expression in kidneys in the basal state was partially IRF-1–dependent, and it will be of interest to identify this additional control mechanism and its relationship to the IFN-{gamma}–independent induction of PIV after tissue injury.

Transcriptional activation of the promoter PIV emerges as the crucial step in CIITA induction in response to allostimulation, LPS, and IFN-{gamma} administration. It probably represents the major mechanism of class II induction in nonlymphoid tissues in infections and graft rejection. The massive class II expression that is induced by systemic and local inflammatory stimuli in vivo is also completely dependent on IFN-{gamma} and will probably prove to reflect PIV activation (6,26). Present data also provide evidence that in vivo PIV induction relies mainly on IRF-1, consistent with our previous conclusion that CIITA mRNA and class II protein expression in kidney is largely dependent on IRF-1 (26,28). The residual PIV induction in the IRF-1KO mice after allogeneic stimulation may reflect STAT1{alpha} binding to its cognate sequence in PIV to activate transcription even in the absence of IRF-1. Thus phosphorylated STAT-1 has a substantial contribution to IFN-{gamma}–induced CIITA upregulation (36), although IRF-1 greatly amplifies this effect (18,37). Other IRF family members may partially compensate for IRF-1 deficiency in activating PIV transcription, particularly IRF-2 (38).

The present results indicate that the major mechanism of CIITA and class II induction after renal ischemic injury is IFN-{gamma}–independent activation of PI and PIV. We have previously shown that acute renal ischemic injury induced the expression of MHC class II and CIITA, which persisted in GKO mice, indicating at least partial independence of IFN-{gamma} (6,29). It is possible that the PI mRNA component is in the interstitial cells, due to enhanced infiltration (25), and the PIV component is in the epithelium. Under some circumstances, IFN-{gamma} production by interstitial infiltrating cells may also contribute (6), although we did not see this component in this experiment. We conclude that CIITA PIV promoter can be activated in injured kidneys by an IFN-independent pathway, a response of particular interest in relationship to autoimmunity and allograft rejection. A strong candidate for IFN-independent PIV activation could be the JAK/STAT pathway that is a significant factor in myocardial ischemia (39) and ischemic brain injury (40). Injury-related PI activation, on the other hand, could reflect accumulation of the class II–positive interstitial cells that occurs several days after renal injury. In fact, GKO mice show as many class II–positive interstitial cells after injury as do WT mice (6). Therefore the amount of PI mRNA expressed in the organ can be attributed not only to transcriptional effects, but also to interstitial infiltration (41). One point to keep in mind is that, compared with the MHC induction that accompanies allogeneic stimulation, LPS, or rIFN-{gamma} administration, these injury effects are weak (6).

The present studies in kidney show how the unusual arrangement of three promoters for one gene gives remarkable range of class II expression in cell types and in response to stimuli. This explains how circulating B cells can be strongly class II–positive regardless of their environment, how cells of the dendritic lineage can be strongly class II–positive but very responsive to environmental stimuli, and how epithelial cells can express class II in times of infection, presumably to help recognition of infected cells. Given the key role of class II in CD4 T cell recognition, this sophisticated arrangement may offer a number of advantages that could not be achieved with a single promoter. It will be of interest as genomics advances to see how often this strategy has been employed and for what types of genes.

Given the role of class II - CD4 T cell interactions in autoimmunity and allograft rejection, the specific CIITA promoters could offer targets for selective control of class II expression in various cell types and tissue compartments, possibly separating the functions of class II in the afferent and efferent limbs of T cell recognition. Having confirmed that CIITA promoters operate in kidney as predicted, the next phase will be to establish the functional importance of these components of CIITA expression. Strategies to selectively suspend the functions of each promoter should now be devised, using mutagenesis, inhibitory RNA, and potentially drugs. The fact that kidneys (42) and hearts (43) lacking CIITA show a reduction in the severity of rejection, despite becoming infiltrated and showing pathologic lesions, support our belief that class II expression has functional effects in transplantation. The identification of the individual transcripts could also be useful in monitoring the components of CIITA transcription and thus of class II transcription in disease processes.


   Acknowledgments
 
This research has been supported by operating grants from the Canadian Institutes of Health Research, the Roche Organ Transplant Research Foundation, the Kidney Foundation of Canada, Novartis Pharmaceuticals Canada, Inc., The Muttart Foundation, and The Royal Canadian Legion. Dr Halloran holds a Canada Research Chair in Life Sciences Related to Human Health and Disease.


   Footnotes
 
Oki Takeuchi and Tasha N. Sims contributed equally to this work.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication July 9, 2003. Accepted for publication August 12, 2003.




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