Evaluation of the effects of photodynamic therapy with phosphorus 31 magnetic resonance spectroscopy.

Magnetic resonance spectroscopy in situ was used to study changes in phosphorus 31 metabolism after photodynamic therapy (PDT) of transplanted HeLa cell tumours. Tumours were irradiated 2 h after administration of ATX-S10 (8-formyloximethylidene-7-hydroxy-3-ethenyl-2,7,12,18, tetramethyl-porphyrin-13,17-bispropionil aspartate), a new photosensitizer and chlorin derivative. Nuclear magnetic resonance spectra were measured prior to illumination and 1, 3, 7, 14, 21 and 28 days after PDT on each mouse. A drastic decrease in adenosine triphosphate (ATP) and a concomitant increase in inorganic phosphate (Pi) were evident on the first day after PDT in all cases. The beta-ATP/total phosphate (P) ratio was 0.64 +/- 0.29% (average +/- s.d.) in complete response, 0.67 +/- 0.30% in recurrence and 2.45 +/- 0.93% in partial response. Comparison of this ratio to the histological findings revealed that the beta-ATP/total P ratio reflects the HeLa cell tumours which survived PDT. In other words, partial response on the one hand was distinguished from complete response and recurrence on the other with this ratio 1 day after PDT (P < 0.05). In addition, the ratio of phosphomonoester (PME) to Pi rose beyond 1.0 when macroscopic recurrence occurred, while it stayed under 1.0 in complete response. This finding suggests that the recurrence of HeLa cell tumours can be detected by the PME/Pi ratio.

[Effective mechanisms of ATX-S10, a new photosensitizer, on HeLa tumors in nude mice].

Photodynamic therapy (PDT) is a treatment modality that utilizes a photosensitizing drug activated by laser generated light. PDT is effective for oncologic applications. Many cancer patients have undergone a hematoporphyrin derivative (HpD)-mediated PDT. The HpD showed a side effect causing prolonged cutaneous photosensitivity. But ATX-S10, a new photosensitizer, provides rapid plasma and tissue clearance, comparable photoactivation efficiency, and superior light absorption of visible red. In this study, the tumor rejection mechanisms of PDT using ATX-S10 on HeLa tumors in nude mice were investigated with morphological and fluorometric methods. The mice were intracutaneously inoculated with HeLa cells, 5 x 10(5) or 1 x 10(7) cells. When tumors grew to about 10-12 mm in diameter, mice were intraperitoneally administered ATX-S10, 30 mg/kg, and 2 hours later the ATX-S10 in tumors was indirectly measured by a fluorometric machine, PMA-10 (Hamamatsu Photonics K. K.) and the tumors were irradiated by Optical Parametric Oscillator (Hamamatsu Photonics K. K.) tuned to a wave length at 670 nm, 5 mJ/pulse, 100 J/tumor. Before and after the irradiation, the effective mechanisms of PDT with ATX-S10 were studied by histological and ultrastructural approaches. The results showed occlusive thrombi in the microvasculature of the tumors and tumor cell death. These occlusive thrombi were observed within one hour after PDT at both light and electron microscopy levels, and were more remarkable as time passed after PDT. Therefore, the morphological studies of PDT with ATX-S10 suggested that the rejection mechanisms occurred mainly as a result of the destructive changes of the microvasculature in the tumors first, and secondly or simultaneously, tumor cells were destroyed through necrosis, and finally the tumors were rejected.

Analysis of pathogenesis of autoimmune insulitis in NOD mice: adoptive transfer experiments of insulitis in ILI and NOD nude mice.

In an effort to study the pathophysiological events in the development of insulitis in NOD mice, we have developed ILI- and NOD-nu/nu mice. ILI mice are a nondiabetic inbred strain but are derived from the same JcI:ICR mouse as NOD mice and share the same H-2 allotype with NOD mice. Splenocytes and CD4+ cells from diabetic NOD mice appeared to transfer insulitis to ILI-nu/nu mice, suggesting that ILI mice already express autoantigen(s) responsible for insulitis. But reciprocal thymic grafts from NOD mice into ILI-nu/ nu mice and those from ILI mice into NOD-nu/nu mice failed to allow the development of insulitis, implying that ILI mice possess neither precursor T cells nor the thymic environment responsible for the development of insulitis. In addition, splenocytes from ILI mice appeared to contain regulatory cells which suppress the development of diabetes but not that of insulitis in NOD mice. The use of these nude mice should provide more information on the products of insulitis-susceptibility genes of NOD mice.

A promiscuous T cell hybridoma restricted to various I-A molecules.

In a previous study, we identified T cell receptor and major histocompatibility complex (MHC) contact sites on the pigeon cytochrome c p43-58 peptide. Positions 46 and 54 of p43-58 were shown to be the MHC-binding sites. Specific amino acids were identified on the MHC-binding sites which bound to the relevant I-A molecule. In the present study, using NOD (I-Ag7) mice, we established a T cell hybridoma specific for a p43-58 analog 46R50E54A with arginine (R) and alanine (A) at positions 46 and 54, respectively. Interestingly, NOE 33-1-2 recognized 46R50E54A in the presence of not only I-Ag7, but also I-Ad, s, u and v. In contrast to previous reports that promiscuous T cells were able to recognize peptide antigens with various HLA-DR or I-E molecules consist of monomorphic alpha and polymorphic beta chains, the promiscuous T cell clone NOE33-1-2 recognized peptides with various I-A molecules lacking the monomorphic chain.

Effect of staphylococcal enterotoxin B-induced anergy on cytokine gene expression: anergy-sensitive and resistant mRNA expression.

We examined the effect of staphylococccal enterotoxin B (SEB)-induced anergy on expression of six different cytokine genes in T cells restimulated with SEB in vitro. We found that although IL-2, IL-3, and IL-4 mRNA levels are substantially reduced in anergic T cells, mRNAs for IL-6, IL-10, IFN-gamma, and TNF-alpha are expressed normally. Thus, there appeared both anergy-sensitive and resistant cytokine mRNA expression in restimulated anergic T cells. The same pattern of cytokine mRNA responses was observed in anergic CD4+ T cells, indicating that the preferential induction of anergy in Th1-like cells is not evident in this in vivo model. Employing TCR V beta 8.2 transgenic mice in which almost all T cells become anergic, we found that the TCR/CD3 complex can transduce both anergy-sensitive and resistant signals. Furthermore, a series of experiments using FK506, A23187, and PMA suggests that signals between TCR and activation of calcineurin and protein kinase C may be blocked in anergic T cells. This is supported by our gel mobility shift assays indicating that calcineurin and/or PMA-inducible NF-ATp, OAP40, and AP-1, but not calcineurin-independent Oct-2, are repressed in anergic spleen T cells upon restimulation with SEB. Taken together, these results suggest that, among signals elicited by stimulation of TCR with SEB, a Ca2+/calcineurin-NF-ATp pathway and other signals, including protein kinase C, are repressed in anergic T cells upstream of their activation, which are essential for the cytokine mRNA expression of the anergy-sensitive type but are dispensible for those of the anergy-resistant type.

Unimpaired clearance of Mycobacterium bovis BCG infection in selectively T-cell anergic TCR-V beta 8.2 transgenic mice.

The anergy induced in mice with staphylococcal enterotoxin B (SEB) has been shown to involve selective unresponsiveness in cytokine expression. While interleukin-2 (IL-2), IL-3 and IL-4 mRNA levels are substantially reduced in anergic T cells upon restimulation with SEB, mRNA for interferon-gamma (IFN-gamma) is expressed normally. On the other hand, infection with Nippostrongylus brasiliensis is known to break an established T-cell anergy. This knowledge prompted us to examine the effect of infection with an intracellular microbe, bacillus Calmett-Guérin (BCG), on the expression of anergy induced with SEB. We have demonstrated that while the SEB-induced anergy was not abrogated by BCG infection, the V beta 8.2 transgenic mice, in which almost all T cells were anergized with SEB, were capable of developing the effective acquired protective immunity, possibly through the preserved capacity to induce IFN-gamma leading to induction of nitric oxide synthase.

Dissection of strain difference in acquired protective immunity against Mycobacterium bovis Calmette-Guérin bacillus (BCG). Macrophages regulate the susceptibility through cytokine network and the induction of nitric oxide synthase.

Protection against infection with intracellular pathogens operates in two stages, early innate resistance and late acquired protective immunity (API), in inbred mouse strains. Although both C57BL/10 (B10) and BALB/c mice bear the susceptible phenotype of innate resistance, Calmette-Guérin bacillus (BCG) vaccination generated efficient API in B10 but not in BALB/c mice. Employing a specific nitric oxide (NO) synthase inhibitor, we revealed that NO production plays a pivotal role in the API of B10 mice. Consistent with this, expressions of the inducible isoform of NO synthase (iNOS) protein and mRNA were significantly higher in the spleen of B10 mice than in that of BALB/c mice. Furthermore, IFN-gamma, a potent inducer of iNOS, and mRNAs for IL-12 (p40); an inducer of IFN-gamma and IL-2 were also vigorously expressed in the spleen of B10 mice compared with that of BALB/c mice. In an attempt to clarify the mechanism by which the different capacities for API are generated, we analyzed the cytokine network between T cells and macrophages in both B10 and BALB/c mice. We found that multiple functions of macrophages, which include capacities to express IL-12 (p40) mRNA in response to BCG and to express mRNAs for iNOS and IL-12 (p40) in response to IFN-gamma, were impaired in BALB/c mice as compared with B10 mice. However, T cells appeared to express comparable level of IFN-gamma mRNA in both strains when stimulated with IL-12. Taken together, these results indicate that the macrophage functions play a pivotal role in both the induction and effector phases of API to determine the susceptibility of mice to BCG infection.

Dissection of positive regulatory elements in the upstream region of the HLA-DPB1 gene.

We investigated the cis-acting sequences that function in the B-cell-specific expression of the HLA-DPB1 gene. Class II B major histocompatibility genes contain a conserved upstream sequence that is important in the expression of these genes. This region has been divided into three major elements, the W, X, and Y boxes. In this paper, we identified an additional positive regulatory element upstream from the DPB1 W box. Using 5' deletion mutants and a substitution mutant, we mapped a positive element, called the W' box, between -184 approximately -169 bp. Sequence comparison revealed that the W' box shares homology with the W box. Electrophoretic mobility shift assay confirmed that the W box binds proteins that also recognize the W' box. Furthermore, deletion and substitution mutants indicate that the W and W' boxes effectively enhance CAT activities only when the X and Y boxes exist.

Analysis of junctional sequences of T cell receptor gamma chain transcripts in gamma delta T cells from rheumatoid synovial fluid.

OBJECTIVE: We previously reported the predominance of the V gamma 3 gene in synovial fluid mononuclear cells (SFMC) from patients with rheumatoid arthritis (RA); the V gamma 3 gene is rare in normal peripheral blood mononuclear cells (PBMC). Our objective was to sequence the nucleotide composition of the junctional region of gamma chain transcripts expressed in gamma delta T cells from RA SFMC and normal PBMC. METHODS: cDNA from RA SFMC and normal PBMC were amplified by polymerase chain reaction, and the nucleotide sequences of amplified clones were determined. We compared the frequencies of V gamma-J gamma rearrangements in inframe (functional) and out of frame (nonfunctional) V gamma transcripts to determine the effect of intrinsic gene rearrangement on the expressed repertoire. RESULTS: Most V gamma genes, including V gamma 9, both inframe and out of frame, were rearranged to the J1/J2 genes, indicating that the intrinsic rearrangement may be influential in molding the mature functional gamma delta T cell repertoire. V gamma 9 transcripts in RA SFMC predominantly used J1/J2, as opposed to JP in normal PBMC. Most N regions displayed extensive diversity in both RA SFMC and normal PBMC, but some functional V gamma 9 transcripts of RA SFMC from one patient showed an identical junctional sequence, and 2 functional clones from different RA SFMC showed an identical junctional sequence, implying that the V gamma 9-J1/J2 transcripts could be controlled by antigenic selection. CONCLUSION: These results demonstrate that the prominent occurrence of the J1/J2 genes in all V gamma genes and the oligoclonality of functional V gamma 9 transcripts, in addition to the predominance of the V gamma 3 gene, are striking features of gamma chain transcripts in RA SFMC:

[Restricted T cell receptor V beta gene expression in bronchoalveolar lavage lymphocytes of patients with sarcoidosis].

T lymphocytes in bronchoalveolar lavage (BAL) fluid from patients with sarcoidosis are activated. They are thought to recognize as yet unknown antigens and to play an important role in the pathogenesis of the disease. We studied the use of the T cell receptor V beta gene of lymphocytes obtained by BAL and lymphocytes obtained from peripheral blood of 11 patients with sarcoidosis and 9 normal controls, using the reverse transcriptase-polymerase chain reaction. As compared to the normal controls, V beta 2 and V beta 6 genes were predominantly expressed (> 15% of the sum of all V beta transcripts) on lymphocytes obtained from BAL fluid in 4 and 7 of 11 patients with sarcoidosis, respectively, but no specific V beta gene was predominantly expressed on lymphocytes obtained from peripheral blood. These results imply that those lymphocytes that are obtained from BAL fluid and that express V beta 2 and V beta 6 genes are involved in the pathogenesis of sarcoidosis.

[Analysis of T cell receptor V beta gene expression in bronchoalveolar lavage fluid lymphocytes from patients with idiopathic interstitial pneumonia].

T lymphocytes in bronchoalveolar lavage (BAL) from idiopathic interstitial pneumonia (IIP) patients are considered to recognize unknown antigens, such as dust, fume, virus or degenerated autoantigens. To analyse the nature of these T lymphocytes, we investigated T cell receptor (TCR) V beta gene usage (22 kinds) in BAL Lymphocytes and Peripheral blood lymphocytes from 10 11P patients and 9 normal controls, using reverse transcriptase-polymerase chain reaction method. In BAL lymphocytes, predominant usage of V beta genes (> 15% of the sum of all V beta transcripts) was recognized in 7 of 10 IIP patients, which appeared to vary in individuals (Case 1: V beta 14, case 2: V beta 3, V beta 5.1, case 5: V beta 2, case 6: V beta 6, case 7: V beta 8, case 8: V beta 7, V beta 20, case 9: V beta 6), whereas no predominant V beta usage was demonstrated in normal controls. It remains to be elucidated whether the BAL lymphocytes expressing predominant V beta genes are involved in the activation of alveolar macrophages, fibroblasts, thereby inducing the production of autoantibodies.

Molecular analysis of the pathogenesis of autoimmune insulitis in NOD mice.

Among diabetes-susceptibility genes in NOD mice, only Idd-1 has been clearly assigned: Idd-1 could be a gene complex composed of class II major histocompatibility complex (MHC) genes, I-A beta and I-E. Employing restriction fragment length polymorphism (RFLP) analysis and nucleotide sequencing, we revealed that ILI and CTS mice, which are nondiabetic but are derived from the same Jcl-ICR mice as NOD mice, share the same class II MHC genes with NOD mice suggesting that both ILI and CTS mice also possess susceptible Idd-1 genotype. This was supported by a breeding study. To compare the usage of T cell receptor (TCR) V beta genes in NOD mice with that in ILI mice, we employed quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) which revealed that TCR V beta usages of these mice were indistinguishable. RT-PCR method also revealed that the V beta transcript of T cells infiltrating into pancreas of NOD mice was not restricted but was rather diverse. Since NOD and ILI mice share the same class I and II MHC antigens, we performed lymphocyte transfer experiments between these mice to examine the mechanism by which ILI mice do not develop insulitis. The results of reciprocal transfer of lymphocytes from NOD to ILI-nu/nu mice or from ILI to young NOD mice suggest that ILI mice exhibit autoantigens responsible for the development of insulitis but do not possess T cells reacting with islets. Of the diabetes-susceptibility genes, only in the case of Idd-1 is there any evidence for the identity of the gene products. ILI mice should provide more information on the products of the other diabetes-susceptibility genes of NOD mice.

The biased V gamma gene usage in the synovial fluid of patients with rheumatoid arthritis.

Taking advantage of the reverse transcriptase-polymerase chain reaction (RT-PCR), we have analyzed T cell receptor gamma-chain mRNA of synovial fluid gamma/delta T cells from patients with rheumatoid arthritis (RA) in comparison with those of peripheral blood mononuclear cells (PBMC) from RA patients and healthy individuals. The quantitative RT-PCR method in conjunction with nucleotide sequencing revealed the frequent usage of the V gamma 3 gene segment in RA synovial fluid mononuclear cells (SFMC) (p < 0.01) which in PBMC of healthy individuals occurred rarely. PBMC of most healthy individuals expressed the V gamma 9 gene predominantly (p < 0.01) as expected. However, only half of RA patients showed elevated levels of the V gamma 9 gene expression in their PBMC. The gamma-chain mRNA containing the V gamma 3 gene in RA SFMC showed no conserved junctional sequence (complementarity-determining region 3). To investigate the nature of ligands recognized by the V gamma 3-bearing T cells, we analyzed V gamma gene usage of RA SFMC, RA PBMC, and normal PBMC stimulated with Mycobacterium tuberculosis (MT) or MT plus interleukin-2 since there is mounting evidence of high reactivity of RA SFMC to MT and mycobacterial heat-shock protein 65. However, the V gamma usage appeared to be mostly V gamma 9 in RA SFMC, RA PBMC and normal PBMC. Taken together these results suggest that an as yet unknown antigen(s) (other than MT) might select gamma/delta T cells expressing the V gamma 3 gene in RA SFMC.

IFN-gamma induces IL-12 mRNA expression by a murine macrophage cell line, J774.

Interleukin (IL)-12 is a logical candidate for participation in the differentiation of T helper (Th) 1 cells. IL-12 is produced by macrophages and B cells and induces the production of interferon (IFN)-gamma from Th1 cells and NK cells. In this study, we show that the IFN-gamma itself is capable of inducing IL-12 mRNA expression in murine macrophage cell line J774. The mRNA was apparent by 12h after IFN-gamma treatment, although maximal induction required 36 to 48h. Furthermore, we investigated the signal transduction mechanism responsible for the mRNA expression. The IFN-gamma-inducible IL-12 mRNA was blocked by two protein tyrosine kinase (PTK) inhibitors, genistein and herbimycin A, suggesting that PTK is involved in the IFN-gamma-inducible IL-12 mRNA expression.

Leukotrien B4-induced interleukin-1 beta in synovial cells from patients with rheumatoid arthritis.

We examined the role of Leukotrien B4 (LTB4) in the production of Interleukin-1 beta (IL-1 beta) by rheumatoid synovial cells since a substantial amount of LTB4 has been detected in the synovial fluid from patients with rheumatoid arthritis. The production of IL-1 beta was augmented by LTB4 at concentrations of 10(-9) to 10(-8) M. Furthermore, LTB4 showed the additive effect on the IL-1 beta production with interferon-gamma but not with lipopolysaccharide. These results suggest that LTB4 in cooperation with certain cytokines might play a pivotal role in the IL-1 beta production by rheumatoid synovial cells in vivo.

HLA class II molecule-mediated signal transduction mechanism responsible for the expression of interleukin-1 beta and tumor necrosis factor-alpha genes induced by a staphylococcal superantigen.

Superantigens including staphylococcal enterotoxins (SE) bind to major histocompatibility complex class II molecules and interact with T cells bearing particular V beta chains. SEB was shown to induce the expression of interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha genes in human peripheral blood monocytes bearing HLA class II molecules. Monoclonal antibodies directed against HLA-DR and -DQ abolished the SEB-induced expression of both the IL-1 beta and TNF-alpha genes, suggesting that the HLA class II molecules mediated the gene expression. Therefore, we investigated the signal transduction mechanism responsible for the expression of IL-1 beta and TNF-alpha genes induced by binding of SEB to the HLA class II molecules. Three protein tyrosine kinase (PTK) inhibitors, genistein, herbimycin A, and tyrphostin, each of which has a different mechanism of action, strongly inhibited the expression of the monokine mRNA induced by SEB. Analyses of PTK activity revealed that SEB induced a rapid increase of membrane-associated PTK activity and this was blocked by tyrphostin. Furthermore, H-7 inhibited the expression of the monokine mRNA induced by SEB, suggesting the involvement of protein kinase C (PKC) in the signaling pathway. The involvement of PKC was confirmed by the observations that phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC, induced the expression of the monokine mRNA and that SEB evoked the activation of membrane-associated PKC. Both activation of PKC and expression of the monokine mRNA induced by SEB appeared to be inhibited by tyrphostin, but those induced by PMA were not. Taken together, these findings indicate that both PTK and PKC play essential roles in HLA class II molecule-mediated signal transduction elicited by SEB and that PTK activation may precede PKC activation in the signaling pathway.

Inhibition of tyrosine phosphorylation prevents IFN-gamma-induced HLA-DR molecule expression.

We have previously demonstrated that HLA-DR molecule expression induced by IFN-gamma is associated with phosphatidylinositide turnover, activation of protein kinase C, and elevation of intracellular calcium. Because phosphorylation of phospholipase C-gamma 1 on tyrosine residues is known to be involved in the activation of phosphatidylinositide turnover, we investigated the role of tyrosine protein kinase (TPK) in the signal transduction for IFN-gamma-inducible DR molecule expression on T98G cells. The effects of three specific TPK inhibitors, genistein, herbimycin A, and tyrphostin, suggest that TPK is involved in the signal transduction. These inhibitors inhibited the IFN-gamma-inducible DR molecule expression in a dose-dependent manner. Being consistent with this, immunoblotting with an anti-phosphotyrosine mAb revealed that IFN-gamma induces a rapid increase in protein tyrosine phosphorylation. Genistein not only abrogated the IFN-gamma-induced enhancement of tyrosine phosphorylation, but also inhibited the IFN-gamma-induced production of inositol-4-5-triphosphate and the elevation of intracellular calcium. However, these three TPK inhibitors failed to inhibit the DR molecule expression induced by PMA and A23187. These findings suggest that the tyrosine phosphorylation is an early and critical event that precedes phosphatidylinositide turnover leading to activation of protein kinase C and elevation of intracellular calcium concentration during IFN-gamma-inducible DR molecule expression.

A comparative study on T cell receptor V beta gene usages: spleen cells from the non-obese diabetic (NOD) mouse and its non-diabetic sister strain, the ILI mouse, and infiltrating T cells into pancreata of NOD mice.

We analyzed the usage of T cell receptor (TCR) V beta genes of spleen cells of NOD mice in comparison with those of its non-diabetic sister strain ILI mice which show no insulitis and (ILI x NOD)F1 mice. The quantitative polymerase chain reaction (PCR) method revealed that PCR V beta repertoires of these mice are indistinguishable. This is consistent with our previous observation that ILI mice share the same H-2 class II genes with NOD mice. PCR method also revealed that the V beta transcript of infiltrating T cells into pancreas of NOD mice was not restricted but was rather diverse. The role of TCR repertoire in the development of insulitis was discussed.

A rapid and sensitive method for HLA-DRB1 typing by acridinium-ester-labeled DNA probes.

A rapid and accurate detection of HLA class II antigen is essential for transplantation and for the understanding of disease susceptibility. Recent molecular genetic studies have revealed that the number of class II loci and the number of alleles at these loci are greater than had been previously detected. It is, therefore, of great importance to detect these extensive polymorphisms. A great deal of effort has been made on identification of individual HLA class II specificities at the DNA level, called "DNA typing." What seems to be lacking, however, is handling simplicity. Here we accomplished a simple method for HLA-DRB1 typing based on hybridization of acridinium-ester (AE)-labeled DNA probes to amplified DNA. This method is called hybridization protection assay (HPA). By using 13 AE-labeled probes, 20 homozygous B-cell lines and leukocytes from 80 healthy individuals were typed by HPA. The results were completely consistent with those obtained by polymerase chain reaction--restriction fragment length polymorphism. This method is suitable for mass screening because of its procedural simplicity and swiftness.

[The signal transduction mechanism responsible for interferon-gamma-inducible indoleamine 2,3-dioxygenase (IDO) gene expression in T98G cells].

The interferon (IFN)-gamma-induced indoleamine 2,3-dioxygenase (IDO) is implicated in the inhibition of intracellular pathogens, e.g. Chlamydia psittaci and Toxoplasma gondii. The intracellular signaling molecules responsible for the induction of IDO gene expression were investigated by the quantitative polymerase chain reaction. The gene expression was inhibited by a tyrosine kinase inhibitor, genistein. Being consistent with this, IFN-gamma induced increased tyrosine phosphorylation and this was inhibited by genistein. The transcription of IDO gene was not inhibited by protein kinase C (PKC) inhibitors, H-7 and staurosporine, or a calmodulin inhibitor, W-7. Irrelevance of PKC in IDO gene expression was supported by the failure of PMA or PMA + A23187 to induce IDO gene expression. These results all suggest that the tyrosine phosphorylation is a critical event in IFN-gamma-inducible IDO gene expression and PKC is not involved in the gene expression.