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Expression of Thrombospondin in TGF-Treated APCs and Its Relevance to Their Immune Deviation-Promoting Properties¹

Department of Ophthalmology, Schepens Eye Research Institute, Harvard Medical School, Boston, MA 02114

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
APCs deployed within iris/ciliary body are responsible for promoting anterior chamber-associated immune deviation following injection of Ag into the eye. TGF-2, a constituent of the ocular microenvironment, converts conventional APCs that are pulsed with Ag into cells that induce immune deviation when injected into naive mice. TGF-2-treated APCs under-express IL-12 and CD40, and over-express active TGF. We have examined transcriptional changes within macrophage hybridoma no. 59, which promotes Th1 cell differentiation, and TGF-2-treated no. 59 as well as macrophage hybridoma no. 63, both of which induce immune deviation similar to anterior chamber-associated immune deviation. Immune deviation-inducing hybridomas up-regulated expression of thrombospondin, TGF, IFN- and , murine macrophage elastase, and macrophage-inflammatory protein-2 genes, while down-regulating expression of the genes for NF-B and CD40. Based on the known properties of these gene products, a model is proposed in which these gene products, alone and through interacting signaling pathways, confer upon conventional APCs the capacity to create and surround themselves with an immunomodulatory microenvironment. The model proposes that the pleiotropic effects of thrombospondin are primarily responsible for creating this microenvironment that is stabile, rich in active TGF and IFN- and , deficient in IL-12, and chemoattractant via macrophage-inflammatory protein-2 for NK T cells. It is further proposed that presentation of Ag to T cells in this microenvironment leads to their differentiation into regulatory cells that suppress Th1 cell-dependent immunogenic inflammation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immune privilege, an important physiologic feature of the eye, is dependent in part upon the ability of the eye to promote systemic tolerance of eye-derived Ags. Injection of Ags into the anterior chamber, vitreous cavity, and subretinal space of the eye routinely elicits a deviant systemic immune response that is deficient in T cells that mediate delayed hypersensitivity, and Abs that fix complement (1, 2, 3). This stereotypic modification of the immune response to eye-derived Ags has been termed anterior chamber-associated immune deviation (ACAID).⁴ It has been argued that ocular immune privilege and ACAID are evolutionarily determined mechanisms that protect the visual axis of the eye from the blinding effects of intraocular inflammation. Understanding the cellular and molecular bases of ACAID has been a central goal of our laboratory for more than two decades.

Based on direct and indirect experimental evidence, it is believed that ACAID arises when bone marrow-derived dendritic cells and macrophages that are indigenous to the eye come under the immunomodulatory influences of the ocular microenvironment. Chiefly, TGF-2, a major constituent of normal aqueous humor, has been implicated in conferring upon indigenous ocular APCs the capacity to promote ACAID (4). Under the influence of TGF-2, intraocular APCs capture Ags, migrate through the trabecular meshwork directly into the blood vasculature, and traffic to the marginal zone of the spleen where they generate the unique spectrum of T and B lymphocytes found in ACAID (5, 6, 7). In this process, eye-derived APCs secrete chemokines that recruit NK T cells to the marginal zone. Together, the APCs and NK T cells establish a unique microenvironment that further attracts B lymphocytes and, eventually, the CD4⁺ and CD8⁺ T cells that become the regulatory T cells of ACAID (8, 9).

APCs obtained from conventional body sites (peritoneal cavity and spleen) can be converted into ACAID-inducing cells by treatment in vitro with active TGF-2. When these TGF-2-treated cells are pulsed with nominal Ag and then injected i.v. into naive mice, the recipients acquire Ag-specific immune deviation similar to ACAID (4, 10, 11, 12). Our laboratory has extensively characterized the phenotype of TGF-2-treated APCs. The key features that appear to distinguish conventional from TGF-2-treated APCs are that the latter are impaired in their capacity to secrete IL-12 and to express CD40 (13).

Because eye-derived APCs lie at the heart of ACAID induction, we are interested in knowing the molecular mechanisms by which TGF confers ACAID-inducing properties on conventional APCs. To gain insight into this issue, we have turned to differential gene expression between two functionally distinct macrophage hybridomas created by Dorf and colleagues (14, 15) in the 1980s. When pulsed with BSA and injected i.v. into naive mice, macrophage hybridoma no. 63 induces immune deviation similar to ACAID. In contrast, macrophage hybridoma no. 59 that has been pulsed with BSA and similarly injected, promotes conventional BSA-specific immunity (10). More importantly, pretreatment of hybridoma no. 59 with TGF followed by BSA pulsing in vitro endows the cells with the capacity to induce immune deviation when injected into naive mice. This observation was also extended to APCs derivatized with a hapten as opposed to those pulsed with a soluble Ag such as BSA. That is, while untreated hapten-derivatized no. 59 (injected s.c.) induced conventional contact hypersensitivity response, TGF treatment conferred upon them the ability to induce hapten-specific tolerance (16). Thus, treatment of no. 59 in vitro with TGF converts the functional phenotype of the cell to that of no. 63. Using differential display and RT-PCR, we have been able to identify genes whose products bear the capacity to: 1) promote activation of latent TGF and tether the active cytokine to the cell surface (e.g., thrombospondin); 2) bind surface receptors on the hybridomas that transduce signals that inhibit production of IL-12 (e.g., thrombospondin and IFN-); and 3) chemoattract NK T cells (e.g., macrophage-inflammatory protein (MIP)-2). Based on these results, a model is presented which attempts to explain the molecular and genetic mechanisms by which TGF confers ACAID-promoting properties on APCs.

	Materials and Methods

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Mice

C3D2/F₁ (H-2^k/d) mice, 6–8 wk old, were purchased from Jackson Laboratories (Bar Harbor, ME) and used to obtain T cells syngeneic to the macrophage hybridoma cells.

Hybridomas and cell culture

Macrophage hybridoma clone nos. 59 and 63 are fusion products of macrophages harvested from the spleen of a CKB (H-2^k) mouse and P388D1 tumor cell line (H-2^d; Ref. 14). Cells were cultured in complete RPMI 1640 (BioWhittaker, Walkersville, MD) containing 1% HEPES, 1% Penn-Strep, 1% Glutamine, 10% FBS, 1% sodium pyruvate, 1% nonessential amino acids, and 0.1% 2-ME.

TGF treatment

Cells of clone no. 59 were cultured overnight with TGF-2 (R&D Systems, Minneapolis, MN; 5 ng/ml final concentration) in near serum-free culture medium (RPMI 1640) containing 25 mM HEPES, 100 U/ml penicillin, 50 µg/ml streptomycin and glutamine, 1% normal mouse serum, and BSA at a final concentration of 5 mg/ml.

Flow cytometry

Hybridoma cells were analyzed by flow cytometry to assess cell surface expression of various molecules. Fluorochrome (FITC or PE)-conjugated Abs used to stain cells included anti-H-2K^d, anti-IA^d, anti-B7.1, anti-B7.2, anti-CD40, anti-CD47 (BD PharMingen, San Diego, CA), anti-CD36 (Santa Cruz Biotechnology, Santa Cruz, CA), as well as isotype-matched control Abs. Stained cells were washed with PBS containing 1% BSA and analyzed on a Coulter Epics XL flow cytometer and Coulter system II software (Beckman Coulter, Miami, FL). Mean fluorescence intensity was determined for 10,000 events. The significance of each data point was assessed by calculating the half-peak correlation variance.

RNA isolation

mRNA was isolated from cells cultured with and without TGF-2 using the Fast Track 2.0 mRNA Isolation Kit (Invitrogen, San Diego, CA) which uses oligo(dT) cellulose, according to the manufacturer’s instructions provided.

RNA arbitrarily primed PCR (RAP-PCR)

RAP-PCR was performed to fingerprint RNA gene transcripts for differential display analysis according to the instructions provided with reagents obtained from Stratagene (La Jolla, CA). In brief, first-strand synthesis was conducted under low-stringency conditions using a single 18-base arbitrary primer of known sequence, which anneals and extends from sites within the mRNA. Second-strand synthesis proceeded in a manner similar to first-strand synthesis during a single round of low-stringency PCR using a second 18-base arbitrary primer. Performing the initial reverse transcription reaction and the subsequent first round of PCR under conditions of low-stringency (7 min at 94°C, 5 min at 30°C, 5 min at 72°C, and hold at 4°C) results in generating an RNA gene transcript fingerprint. This fingerprint was then amplified in the presence of a radioactively labeled nucleotide by standard high-stringency PCR (5 min at 94°C, 1 min at 94°C, 2 min at 48°C, 3 min at 72°C, 7 min at 72°C, and hold at 4°C for 40 cycles) by virtue of having incorporated arbitrary primers. The resulting PCR product mixture was subsequently analyzed by denaturing PAGE and autoradiography as shown in Fig. 2, and differentially expressed bands were identified.

View larger version (50K): [in this window] [in a new window]   FIGURE 2. RAP-PCR: RAP fingerprints of mRNA from untreated macrophage hybridoma nos. 59, 63, and TGF-2-treated no. 59. mRNA was isolated from untreated hybridoma cells or no. 59 cultured overnight with 5 ng/ml of TGF-2. Each RNA sample (100 ng) was RAP fingerprinted as described in Materials and Methods. [-35S]-labeled PCR products were separated by denaturing PAGE. Two of the differentially amplified products between untreated and TGF-2-treated cells are indicated by arrows. Only a portion of the autoradiogram is shown.

Differentially expressed bands were excised from the gel, which was dried onto 3-mm paper (Whatman, Clifton, NJ) by placing the autoradiogram on the dried gel and cutting through the film. The band was then eluted into 500 µl of water during an overnight incubation at 4°C. A small aliquot (5 µl) of the eluent was used as template for a subsequent PCR using the same primer pairs that originally produced the differentially expressed band. PCR conditions used were the same as those in the high-stringency PCR step of RAP-PCR. Three 50-µl PCR were pooled and subjected to a QIAquick (Qiagen, Valencia, CA) PCR purification procedure for DNA clean-up which removed primer-dimers (partially). The eluent from the QIAquick column was analyzed by agarose gel electrophoresis in the presence of ethidium bromide and showed a single strongly staining band for each differentially expressed gene. The PCR-amplified differentially expressed band was cloned after clean-up with the QIAquick PCR purification kit (Qiagen) using the pGEM-T Vector system (Promega, Madison, WI). After ligation of the insert into the vector, competent JM109 cells (Promega) were transfected and plated on ampicillin plates. Bacteria harboring recombinant plasmids were identified by blue/white screening and a select number of white colonies were used to inoculate 5-ml minicultures. Plasmid DNA was isolated from individual cultures for sequencing using the Qiagen Plasmid mini kit (Qiagen). Plasmid DNA samples were sequenced using the Taq DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). After cycle sequencing and clean-up, DNA samples were analyzed by PAGE on an ABI 377 PRISM-automated sequencer (PE Applied Biosystems, Foster City, CA).

RT-PCR

cDNA was batch synthesized by reverse transcribing mRNA using random hexamers and avian myoblastosis virus reverse transcriptase (Promega). For PCR amplification, cDNAs were amplified using various primers as listed in Table I. Intron spanning primers for the specific amplification of selected genes were designed using gene sequences from the public database and Oligo Primer Analysis software 6.0 (Molecular Biology Insights, Plymouth, MN). PCRs were performed in a 50-µl amplification mixture containing 1x polymerase buffer, 2.5 mM MgCl₂, 0.2 µM each dNTP, 1 µM of forward and reverse primers, 1.25 U Taq polymerase (PerkinElmer, Wellesley, MA). Touchdown PCR analysis with a thermal profile of -94°C for 1 min followed by 70–55°C at two cycles per degree for 2 min and 72°C for 3 min was performed in a thermal cycler (GeneAmp PCR System 2400; PerkinElmer). After 30 thermal cycle amplification, the PCR products were separated by 1.5% agarose gel electrophoresis.

Band densitometry. PCRs were set up as described. Amplification was accomplished at appropriate optimum temperatures for various primers and terminated at various cycles as indicated in the figures. Reaction products were detected using a 1.5% agarose/Tris-acetate-EDTA gel containing 0.5 µg/ml ethidium bromide. Densitometric measurements of bands were used to calculate a ratio of the gene of interest, GAPDH.

Real-time PCR assay. TaqMan primers and probes for real-time PCRassay were designed using Primer Express 1.0 software (Applied Biosystems) and optimum concentrations for each were determined. Theirsequences are as follows: forward primer, 5'-GACTTGCTGCATTTCCGCAT3'; reverse primer, 5'-GCTCAAGTGCCTGTGTGGAAG-3'; probe, carboxyfluorescein-CTGCTGGTGAGAGCTGATTGACCCAA-carboxytetramethylrhodamine. Efficiency of target gene (thrombospondin) and the internal control gene (GAPDH) amplification was compared and confirmed to be equal. Amplification reactions were set up according to the manufacturer’s instructions included with reagents (Applied Biosystems) briefly—each reaction contained 1x TaqMan Buffer A; 200 µM of dATP, dGTP, and dCTP; 400 µM of dUTP, 5.5 mM MgCl₂; 0.01 U/µl uracil N-glycosylase; 900-nM forward and 300-nM reverse primers; 100-nM dual-labeled (FAM-TAMRA) probe, and 5 µl 1/25 dilution of cDNA. The reactions were performed in MicroAmp 96-well plate capped with MicroAmp optical caps and amplified for 40 cycles in Gene Amp 5700 with the standard PCR parameters (thermal profile: 50°C for 2 min, 1 cycle; 95°C for 10 min, 1 cycle; 95°C for 15 s, 40 cycles; 60°C for 1 min, 1 cycle). The data generated from reactions were analyzed by plotting Rn fluorescence signal vs the cycle number. An arbitrary threshold was set at the midpoint of the log Rn (difference between Rn (normalized reporter) values of PCR with template and that without template) vs cycle number plot. The threshold cycle values calculated from this plot were used to determine fold change in target gene and control gene by converting them from logarithmic to linear value. Fold change = 2^-Ct.

In vitro stimulation assays

Hybridoma cells (10⁵/well) were primed with IFN- (BD PharMingen, 1000 U/ml) in a total volume of 100 µl of serum-free RPMI 1640, for 48 h at 37°C. Culture supernatants were replaced with fresh medium containing TGF-2 (5 ng/ml). After overnight incubation, cells were stimulated with anti-CD40 (BD PharMingen) -coated ImmunoPure agarose beads (Pierce, Rockford, IL; 2%) for 48 h. Culture supernatants were collected to examine levels of IL-12 by ELISA. In some experiments, hybridoma cells were cocultured with equal numbers of T cells isolated from OVA-primed mice in the presence of OVA (50 µg/ml). Culture supernatants collected at various time intervals were then tested by ELISA for IL-12. Effect of thrombospondin and IFN- (Calbiochem, La Jolla, CA) on the secretion of IL-12 was assessed by including these reagents at indicated concentrations during CD40-mediated stimulation.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Constitutive expression of cell surface MHC and costimulatory molecules on macrophage hybridomas

Although it is known that hybridoma no. 59 can function as a conventional APC and that no. 63 induces Ag-specific tolerance, it is not known whether expression of cell surface molecules reflects their functional differences. Major histocompatibility and costimulatory molecules are well recognized as cell surface molecules necessary for effective Ag presentation by an APC. Earlier studies conducted on these hybridoma clones compared expression of some cell surface molecules by radioimmunoassays and reported comparable expression of MHC- and macrophage-specific markers and absence of, then available, B and T cell-specific markers (15). In this report we have performed flow cytometric analysis to compare expression of molecules now known to be critical for efficient Ag presentation by an APC. Cells of both hybridomas, nos. 59 and 63, were examined for surface expression of costimulatory molecules B7-1, B7-2, and CD40, as well as MHC class I and II molecules. As revealed in Fig. 1A, both hybridomas were strongly positive for class I molecules, and comparably weakly positive for class II molecules. Expression of costimulatory molecules B7-1, B7-2, and CD40 was found to be comparable on both hybridomas (Fig. 1B).

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Cell surface expression of MHC and costimulatory molecules on macrophage hybridoma nos. 59 and 63. Cells stained with FITC or PE-conjugated Abs against cell surface molecules were analyzed by flow cytometry. A, MHC molecules (class I-anti-H-2Kd, class II-anti-I-Ad): filled histograms represent positively stained cells and open histograms represent staining with isotype-control Ab. B, Costimulatory molecules (B7-1, B7-2, and CD40): solid dark lines and dotted light lines represent staining with specific Ab and isotype-control Ab, respectively.

Given the overall similarity in MHC and costimulation molecule expression by hybridomas no. 59 and 63, in comparison to the profound functional difference between these two hybridomas, it was necessary to attempt alternative approaches to unraveling the molecular basis for the difference.

Comparison of genetic program of APCs by differential display (RAP-PCR)

Differential display or RAP-PCR is a well-known RNA fingerprinting method that allows semiquantitative comparison of the abundances of thousands of mRNAs (17, 18, 19). This method was used to compare the genetic programs of these macrophage hybridomas to determine which genes are selectively expressed or repressed in APCs that induce Ag-specific tolerance or ACAID (hybridoma no. 63 or TGF-treated no. 59).

Macrophage hybridomas were grown in near serum-free cultures as described in Materials and Methods. Hybridoma no. 59 was also subjected to overnight TGF treatment, a treatment that renders their APC function similar to that of no. 63. mRNAs isolated from untreated nos. 59, 63, and TGF-treated no. 59 were then used to generate RNA fingerprints in a RAP-PCR using a pair of random primers, each 18 bases in length. Fifteen such random primers were used in various combinations, which resulted in a total of 105 primer pairs. In each PCR experiment, five pairs were included. Thus, 21 RAP-PCR experiments were completed to allow the use of all 105 pairs. All PCR products were resolved on denaturing polyacrylamide gels as described in Materials and Methods. In Fig. 2, a small portion of one such gel is shown as an example, with differentially amplified gene products indicated by arrows.

Identification of differentially expressed genes isolated by RAP-PCR

Differentially expressed PCR products were further characterized by isolating them from the gel followed by cloning into plasmid vectors. Six clones for each differentially expressed gene were generated. Plasmid DNA for each clone was isolated, purified, and sequenced. All the sequences were then compared with GeneBank DNA sequences using the computer search program NCBI Blast (National Library of Medicine). Identity of an isolated differentially expressed gene was established when sequences of at least two clones matched with the sequence of the same gene in the public database.

Our goal was to identify genes up- and down-regulated in no. 59 in response to TGF treatment. To reach this goal, we compared differentially expressed genes in no. 59, in TGF-treated nos. 59 and 63—APCs that constitutively induced ACAID. Differentially expressed genes were then placed into three categories: 1) up-regulated in ACAID-inducing APCs (no. 63 and TGF-treated no. 59), but not in conventional APCs (no. 59); 2) down-regulated in ACAID-inducing APCs, but not in conventional APCs; and 3) up- or down-regulated in patterns not correlated with ACAID-inducing properties. In subsequent studies, only categories 1 and 2 were considered for further analysis. This classification was based on the capacity of TGF to confer ACAID-promoting properties on conventional APCs. Expression of genes in ACAID/tolerance-inducing hybridoma no. 63 was used as a reference. When TGF-dependent changes in expression levels of certain genes in hybridoma no. 59 were found to be comparable to expression levels of those genes in no. 63, these changes were recorded as either up- or down-regulation of the relevant genes. In other words, comparison was made of the transcripts in hybridoma no. 59 cells that were altered by TGF treatment with the constitutive transcripts present in hybridoma no. 63 cells—a reference hybridoma that spontaneously induces ACAID when pulsed with Ag and injected into naive mice. Only those transcripts found to have similar expression in both no. 63 and TGF-treated no. 59 were selected for further consideration.

A total of 68 differentially expressed transcripts were isolated. Sequences of 72% (n = 49) of these transcripts showed homology with known genes in the public database, while 24% (n = 16) represented genes of as yet unknown identity (three differentially expressed transcripts could not be cloned). Genes with known identities were then divided into three categories as described above. Although 11 genes could be classified as up-regulated (category 1), eight genes were found down-regulated (category 2). The rest (n = 30) did not fit any of the two chosen categories, and were grouped under category 3. Table II lists these classified genes.

To confirm that the observed differences in RAP-PCR experiments correlated with differences in steady-state levels of the corresponding mRNAs, the expression patterns of selected genes were determined by semiquantitative RT-PCR using primers specific for those genes. Reverse transcribed mRNA samples were compared for the levels of specifically amplified product at various numbers of cycles using either the band densitometry or real-time PCR method. Normalizing the levels of selected genes with that of a housekeeping gene, such as GAPDH, allowed for the relative semiquantitative comparison of these genes in each sample. Use of the real-time PCR detection method allowed determination of fold change in mRNA expression after TGF-2 treatment. Either or both of these methods confirmed differential expression of genes in the expected manner. As shown in Fig. 3, expression patterns of tested genes (murine macrophage elastase, CD40, NF-B, and thrombospondin) matched that detected in RAP-PCR analysis. That is, expression of MME and thrombospondin was found to be up-regulated after TGF-2 treatment (Fig. 3, A and B), whereas that of CD40 and NF-B was found to be down-regulated (Fig. 3, C and D). In addition, up-regulation of thrombospondin mRNA was confirmed by northern blot analysis (data not shown).

View larger version (35K): [in this window] [in a new window]   FIGURE 3. Confirmation of genes differentially expressed upon TGF-2 treatment of macrophage hybridoma no. 59. mRNA samples from untreated and TGF-2-treated no. 59 were tested for the expression of A, MME; B, CD40; C, NF-B; and D and E, TSP using semiquantitative RT-PCR or real-time PCR. The results obtained by band densitometry method (A–C) are represented as ratios relative to the levels of housekeeping gene GAPDH. Real-time PCR analysis of TSP expression (plot of the Rn vs cycle number) is shown in D. The difference in the mRNA levels for TSP and GAPDH are shown in E as relative fold change (values were calculated by converting individual Ct values from a total of four cDNA amplifications to a linear value equal to 2-Ct).

Although RAP-PCR allowed comparison of changes in the abundance of anonymous RNAs that may reflect changes in the functional properties of APCs, we also used RT-PCR in our second approach toward identifying differentially expressed genes. Using this approach, we compared expression of genes that are known to be relevant in APCs involved in immune responses. These genes include inflammatory and anti-inflammatory cytokines, besides the factors that represent the innate immune response. Genes for inflammatory cytokines TNF-, IL-1, IL-1, IL-1Ra, and IL-6 for anti-inflammatory cytokines such as TGF-1 and for complement factors C3 and C5 were found to be differentially expressed in the tested hybridomas (Fig. 4). However, their expression pattern did not fit the chosen criteria of either up-regulated or down-regulated genes as described earlier. No message for cytokines IL-10, GM-CSF, IL-12 (p40), or IFN- was detected in any of the hybridomas (data not shown). However, type I IFNs (IFN- and IFN-) were differentially expressed and matched the chosen criteria for the up-regulated genes. That is, message for these cytokines was up-regulated in hybridoma no. 59 after TGF treatment resembling that found in untreated hybridoma no. 63, compared with untreated hybridoma no. 59, suggesting that these cytokines might endow functional similarities upon macrophage no.63 and TGF-treated no. 59.

View larger version (57K): [in this window] [in a new window]   FIGURE 4. Expression pattern in macrophage hybridomas of inflammatory and anti-inflammatory cytokines and factors representing innate immune response. mRNA isolated from untreated nos. 59, 63, and TGF-2-treated no. 59 was subjected to RT-PCR analysis using pairs of specific primers for the listed cytokines, complement components, and housekeeping gene GAPDH. PCR products were analyzed by ethidium bromide/agarose gel electorphoresis.

Differential ability of IFN--primed macrophages to secrete IL-12

Although RT-PCR failed to show any detectable message for IL-12 in any of the macrophage hybridomas, differentially expressed genes detected by RAP-PCR suggested a possibility of differential regulation of IL-12. This cytokine has been demonstrated to play a significant role in the induction of Ag-specific immune response in vivo, as neutralization of this cytokine results in Ag-specific unresponsiveness or tolerance (20). Also, impaired Th1 cell development following Ag injection into the anterior chamber of the eye has been attributed recently to a local deficiency of IL-12 (21). Although the effect of TGF on the ability of certain APCs to secrete IL-12 is known (22, 23), the ability of the macrophage hybridomas used in this study to secrete IL-12 has not been reported. The results of our differential display analysis strongly suggest that functionally similar hybridoma no. 63 and TGF-treated no. 59 are likely to be deficient in their ability to produce IL-12. Thus, TGF may be responsible for altering the genetic program of no. 59 such that pathways to IL-12 production are impaired. These changes may resemble the transcriptional program found constitutively in hybridoma no. 63, which is incapable of inducing an Ag-specific Th1-type immune response. Secretion of IL-12 by macrophages is known to be induced by T cell-dependent CD40-mediated stimuli (24, 25). To test the ability of macrophage hybridomas to respond to such IL-12-inducing stimuli, we first primed macrophages with IFN- and then stimulated them with anti-CD40 Ab or cocultured Ag-pulsed hybridomas with Ag-primed T cells, as described in Materials and Methods. Culture supernatantnts were then tested by ELISA for the levels of secreted IL-12. In comparison to macrophage hybridoma no. 59 (which secreted easily detectable IL-12 in response to stimulation), hybridoma no. 63 failed to produce detectable levels of IL-12 in response to CD40 ligation (Fig. 5, A and B). These results are consistent with recent findings reported by van Seventer and colleagues (26), where IL-12 secretion by human dendritic cells was noted to be regulated by IFN- in that this cytokine inhibited T cell-dependent, CD40-induced dendritic cell production of IL-12. Thus, consistent with the observed profile of differentially expressed genes that included message for IFN- found in ACAID-promoting APCs, down-regulated IL-12 secretion in response to a CD40-mediated stimulus was noted in the ACAID or tolerance-inducing APCs, such as hybridoma no. 63 and TGF-treated no. 59, as compared with untreated no. 59. Furthermore, when a CD40-mediated stimulus was provided in the presence of gene products of up-regulated genes such as thrombospondin and/or IFN-, hybridoma no. 59 failed to secrete detectable levels of IL-12 (Fig. 5C). These findings are consistent with the inhibitory influence of thrombospondin on IL-12 secretion by Staphylococcus aureus Cowan I bacteria- and IFN--stimulated peripheral blood monocytes reported by Armant et al. (27).

View larger version (13K): [in this window] [in a new window]   FIGURE 5. Differential ability of IFN--primed macrophage hybridomas to secrete IL-12. A, In response to CD40-mediated stimuli: IFN- (1000 U/ml)-primed macrophages were cultured overnight with (no. 59) or without TGF-2 (nos. 59 and 63) in serum-free culture medium. Cells were further stimulated with anti-CD40-coated agarose beads (1–2%) for 48 h. Culture supernatants were collected and measured for secreted IL-12 by ELISA; B, In response to T cell-mediated stimuli: OVA-pulsed hybridomas were cocultured with OVA-primed T cells. Culture supernatants collected at various time intervals were tested for the presence of IL-12; C, In the presence of thrombospondin and/or IFN-: IFN--primed no. 59 were cultured with CD40 or isotype Ab-coated beads in presence of medium alone or thrombospondin (1.7 µg/ml) and/or IFN- (5 ng/ml) for 48 h before testing culture supernatants for IL-12.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our previous in vitro studies of the T cell activating potentials of conventional and TGF-2-treated APCs had focused our attention on IL-12 and CD40 expression as critical (13). Specifically, several lines of evidence indicated that TGF-2-treated APCs that promote ACAID in vivo display in vitro a selective, quantitative deficit in their ability to secrete IL-12 and express CD40 (we have preliminary evidence that TGF-treated no. 59 cells express reduced surface levels of CD40, data not shown). At the same time, TGF-2-treated APCs display an enhanced capacity to secrete active TGF (28). Several of the genes we have now found to be differentially expressed in hybridomas no. 59, 63, and TGF-2-treated no. 59 are prime candidates for mediating these effects of TGF-2 on APCs.

With regard to TGF-dependent genes that can influence IL-12 and CD40 gene expression, IFN-, IFN-, and NF-B are prominently represented among the differentially expressed genes in the macrophage hybridomas we studied. NF-B was down-regulated in hybridomas no. 63 and TGF-treated no. 59 compared with untreated no. 59. This result is consistent with the well-characterized role of this NF in promoting inflammatory cytokines as well as IL-12 (29, 30). In contrast, the genes for type I IFNs (IFN- and -) were up-regulated in no. 63 and TGF-2-treated no. 59. These cytokines have been demonstrated to have a broad range of immunomodulatory effects which include down-regulation of IFN--induced MHC class II expression, Th1 cell differentiation, and more recently, IL-12 secretion (26, 31, 32). Besides these properties, recently demonstrated ability of type I IFNs to maintain anergic CD4 T cells may be of significance (33). In addition, the gene for thrombospondin-1 (TSP) was up-regulated in no. 63 and TGF-2-treated no. 59, compared with untreated no. 59. Thrombospondin, through its binding to CD47, has been shown to inhibit expression of IL-12 (27). Also, thrombospondin has been repeatedly shown to promote activation of latent TGF (34, 35, 36). Therefore, enhanced expression of thrombospondin may also be correlated to enhanced activation of TGF, produced by APCs. This active TGF itself, in an autocrine fashion, can continue to inhibit NF-B and IL-12 expression. It is possible that other differentially expressed genes discovered in our search may have similar properties to IFN- and -, TSP, and TGF. Thus, a coterie of genes differentially regulated in macrophage hybridoma no. 59 by TGF-2 inhibits IL-12 and CD40 expression, thereby robbing the cells of the capacity to promote the differentiation of responding T cells down the Th1 pathway.

In prior studies, we reported that neutralization of active TGF restores the ability of TGF-2-treated APCs to activate naive T cells in a manner that leads to Th1 generation (37). Therefore, active TGF is central to the ACAID-inducing properties of TGF-2-treated APCs. Our laboratory has previously reported that TGF-2-treated APCs not only increase their endogenous capacity to secrete TGF, but the cells display an enhanced capacity to convert latent TGF into its active form (28). Examination of the differentially expressed genes among hybridomas no. 59, 63, and TGF-2-treated no. 59 reveals candidate genes that could be involved in enhancing secretion of active TGF by no. 63 and TGF-2-treated no. 59. Thrombospondin stands out particularly as a candidate gene in this regard. TSP is a multifunctional matricellular protein with extensively studied properties that are relevant to TGF activation. First, TSP can bind latent TGF, and this TSP-TGF complex can bind to the macrophage surface via CD36 (36). TSP also has the capacity to bind to surface molecule CD47 expressed on most cells (27, 38). In this manner, TSP can be imagined to "tether" latent TGF within the plane of the APC surface membrane and to promote its cleavage into the active molecule. Thus, the released and activated TGF is concentrated at the surface of the APC. Of equal importance is the fact that T cells and NK T cells also express CD47 constitutively, which makes it possible for thrombospondin to form a molecular bridge between these cells and APCs. In this manner, active TGF can be visualized to be concentrated at the apposed surfaces of T and NK T cells with APC. It may be relevant that TSP was demonstrated recently to be a potent inhibitor of TCR-mediated T cell activation (39). Also, active TGF is known to have potent immunomodulatory effects on T cells, including the activation of regulatory cells, and TGF is important in dictating the functional properties of NK T cells (40, 41). It is attractive to speculate that TGF-2-treated APCs use thrombospondin to orchestrate the creation of an ad hoc microenvironment highly enriched for active TGF. The observation that TSP can bind to CD36 and CD47 on macrophages and consequently lead to increased expression of the TGF gene emphasizes that thrombospondin may be central to ACAID induction.

It was gratifying to discover that macrophage hybridomas no. 63 and TGF-2-treated no. 59 up-regulated expression of MIP-2. Sonoda et al. (8) have recently reported that NK T cells accumulate in the spleens of mice following injection of Ag into the anterior chamber of the eye. These authors further reported that mice deficient in NK T cells were incapable of acquiring ACAID following injection of Ag into the anterior chamber. Subsequently, Faunce et al. (9) have demonstrated that F4/80⁺ leukocytes expressing the MIP-2 gene appear in the blood shortly after injection of Ag into the anterior chamber of the eye. Eventually, significant numbers of MIP-2-expressing F4/80⁺ cells congregate in the splenic marginal zone where NK T cells are subsequently recruited. Mice deficient in MIP-2 are unable to develop ACAID, implying that APCs attract NK T cells to the spleen via secretion of MIP-2. Our finding that treatment of hybridoma no. 59 with TGF-2 up-regulates the MIP-2 gene links this effect directly to the in vivo situation and supports the view that the genes found to be differentially regulated in hybridoma no. 59 treated in vitro with TGF-2 are likely to be operative in vivo.

It is worth commenting on the expression in macrophage hybridomas no. 59 and 63 of genes normally thought to be critically important in presentation of Ag to T cells: MHC class I and II molecules, and potent costimulatory molecules, such as B7-1, B7-2, and CD40. Although these hybridomas are functionally different, they expressed similar constitutive levels of MHC molecules and costimulation molecules. After treatment of these two hybridomas with IFN-, we observed comparable up-regulation of class I. However, this treatment was less efficient at up-regulating class II expression in no. 63 compared with no. 59, but was nonetheless observed in both. The latter result can be due to increased expression of type I IFNs noted in no. 63, as these cytokines are known to influence IFN--mediated MHC class II expression (31). From our results, it appears that expression of class I and costimulatory molecules (B7-1, B7-2) on APCs does not play a decisive role in determining whether the cells will promote sensitization or immune deviation. CD40 is, however, a different matter. Although cell surface expression of this costimulatory molecule appeared slightly reduced in hybridoma no. 63, the message for CD40 was clearly reduced compared with hybridoma no. 59. Furthermore, TGF-2 treatment of no. 59 resulted in decrease in the message levels for CD40. This observation is consistent with our previous finding with respect to the inability of TGF-2-treated peritoneal macrophages to stimulate T cells to secrete IFN- in vitro due to their inadequate CD40 expression. Moreover, TGF-2-treatment of peritoneal macrophages interferes with CD40 expression (13). Our experiments with macrophage hybridomas stimulated with anti-CD40 Abs help to shed light on this situation. Whereas stimulation of hybridoma no. 59 with anti-CD40 Abs caused intense IL-12 production, similar stimulation of hybridomas no. 63 and TGF-2-treated no. 59 led to far less or no IL-12 production. We suspect that treatment with TGF interferes directly (by lowering expression of CD40 or NF-B) or indirectly (by increasing expression of molecules like TSP, IFN- and -) with the ability of macrophage hybridoma no. 59 to respond to IFN- in a manner that permits them to activate Th1 cells.

As mentioned previously, the labor-intensive analysis of RAP-PCR has permitted us to sample 50% of the expressed genes in macrophage hybridomas no. 63 and 59. There are doubtless other important genes yet to be identified, and we are now in the process of using DNA microarrays to reach this goal. But even with an incomplete set of information concerning differentially expressed genes in our macrophage hybridomas, it is already possible to create a model that can be tested experimentally (Fig. 6). We propose that macrophages/APCs treated with active TGF-2 up-regulate expression of the TSP gene. By binding to CD47, TSP signals the APC to decrease expression of IL-12 and by binding CD36 and latent TGF, TSP promotes the accumulation of active TGF at the APC surface. Active TGF in an autocrine manner causes the APC to exaggerate production of TGF and to further inhibit IL-12 expression. Enhanced expression of type I IFNs combined with lowered expression of CD40 and NF-B also contribute toward impaired IL-12 expression. Thus, with grossly impaired IL-12 secretion, APCs are poorly able to activate T cells in a manner that leads to IFN- secretion, and this in turn hampers their ability to activate their own CD40 gene. Simultaneously, adjacent T cells are exposed to active TGF and TSP as their TCR for Ag recognize peptide-MHC complexes on the APC surface. Not only can ligation of CD47 negatively regulate T cell activation, but in the absence of IL-12 and CD40, Tcr-mediated signals lead to a functional program that is devoid of IFN- and effector function. Perhaps this is the genesis of the T cells that have been found to regulate immunogenic inflammation in animals with ACAID.

View larger version (32K): [in this window] [in a new window]   FIGURE 6. Proposed model indicating possible interplay of molecules that lead to down-regulation of IL-12 in TGF-exposed APCs that are known to acquire the ability to induce Ag-specific tolerance.

	Footnotes

 
¹ This research was supported by Proctor and Gamble (International Program for Animal Alternatives) and National Institute of Health Grant EY05678.

² Current address: Transgenomic Incorporated, San Jose, CA 95131.

³ Address correspondence and reprint requests to Dr. J. Wayne Streilein, Department of Opthalmology, Schepens Eye Research Institute, Harvard Medical School, 20 Staniford Street, Boston, MA 02114. E-mail address: waynes{at}vision.eri.harvard.edu

⁴ Abbreviations used in this paper: ACAID, anterior chamber-associated immune deviation; MIP, macrophage-inflammatory protein; RAP-PCR, RNA arbitrarily primed PCR; TSP, thrombospondin-1.

Received for publication September 25, 2001. Accepted for publication January 2, 2002.

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