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The Journal of Immunology, 1998, 161: 3589-3595.
Copyright © 1998 by The American Association of Immunologists

Characterization of a Peptide Analog of a Determinant of Type II Collagen That Suppresses Collagen-Induced Arthritis1

Linda K. Myers2,*, Bo Tang{dagger}, E. F. Rosloniec*,{dagger}, J. M. Stuart*,{dagger}, T. M. Chiang{dagger} and A. H. Kang*,{dagger}

* Departments of Pediatrics and Medicine, University of Tennessee, Memphis, TN 38163; and {dagger} Research Service of the Veterans Administration Medical Center, Memphis, TN 38163


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Immunization of susceptible strains of mice with type II collagen (CII) elicits an autoimmune arthritis known as collagen-induced arthritis (CIA). One analogue peptide of the immunodominant T cell determinant, A9 (CII245–270 (I260->A, A261->B, F263->N)), was previously shown to induce a profound suppression of CIA when coadministered at the time of immunization with CII. In the present study, A9 peptide was administered i.p., orally, intranasally, or i.v. 2 to 4 wk following CII immunization. We found that arthritis was significantly suppressed even when A9 was administered after disease was induced. To determine the mechanism of action of A9, cytokine responses to A9 and wild-type peptide A2 by CII-sensitized spleen cells were compared. An increase in IL-4 and IL-10, but not in IFN-{gamma}, was found in A9 culture supernatants. Additionally, cells obtained from A9-immunized mice produced higher amounts of IL-4 and IL-10 when cultured with CII compared with cells obtained from mice immunized with A2, which produced predominantly IFN-{gamma}. Suppression of arthritis could be transferred to naive mice using A9-immune splenocytes. Lastly, phosphorylation of TCR{zeta} was not altered in the immunoprecipitates from the lysates of cells exposed to analogue peptides (A9 and A10) together with wild-type A2 in a T cell line and two I-Aq-restricted, CII-specific T hybridomas. We conclude that analogue peptide A9 is effective in suppressing established CIA by inducing T cells to produce a Th2 cytokine pattern in response to CII.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Collagen-induced arthritis (CIA)3 is an experimental model of autoimmune-mediated polyarthritis that shares important clinical, histologic, and immunologic features with human rheumatoid arthritis. CIA can be induced in susceptible strains of mice by immunization with type II collagen (CII), the major constituent protein of articular cartilage (1). In mice, susceptibility is linked to the MHC and is limited to animals expressing I-Aq or I-Ar (2, 3). We have previously shown that of the major peptides generated by cyanogen bromide (CB) digestion of CII, only CB11 (CII124–402) contains structural determinants sufficient for the induction of arthritis or for the induction of tolerance in the DBA/1 (H-2q) mouse (4, 5). We have identified four T cell epitopes within CB11 using proliferation and cytokine assays, one of which, CII260–270 is immunodominant (6, 7). Based on these data, we have generated a synthetic analogue peptide, A9, (CII245–270 (I260->A, A261->B, F263->N), containing three amino acid substitutions, that can suppress the initiation of arthritis when given to DBA/1 mice at the time of immunization with CII (8).

In the present paper we show that A9 down-regulates established disease even when given 2 to 4 wk after the induction of arthritis by immunization with CII. Although the mechanism by which this analogue peptide affects arthritis is not completely understood, our hypothesis is that the inhibition is mediated through a TCR-based phenomenon. Because of the amino acid substitutions, the affinity of peptide binding to either the MHC class II molecule or the TCR recognition site of CII260–270-specific T cells may have been altered. One possibility is that by means of an alteration in signaling through the TCR, the analogue may induce T cells to produce an altered profile of cytokines, the majority of which are suppressive-type cytokines capable of down-regulating the production of inflammatory cytokines and autoantibodies that induce and exacerbate autoimmune arthritis. Alternatively, the analogue may function as a TCR antagonist or a partial agonist, either severely inhibiting T cells that ordinarily react with CII260–270, causing them to become completely anergic and refractory to subsequent stimulation, or causing only partial, ineffective signaling through the TCR. A third possibility would be that A9 is a high affinity MHC binding peptide that causes inhibition of T cell activation by blocking the Ag binding site of MHC molecules.

In this study we explore the biochemical events representing the earliest stages of the known signaling pathways in T cells, the array of cytokines produced in response to A9, as well as the binding characteristics of the A9 analogue peptides to the I-Aq molecule to obtain information on the mechanism of suppression of arthritis mediated through this analogue peptide. The results presented in this paper suggest that A9 peptide inhibition is mediated through its ability to induce a Th2-type cytokine profile.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of CII

Native CII was solubilized from bovine articular cartilage by limited pepsin digestion and purified as described previously (9).

Chemical synthesis of oligopeptides of CII245–270

The peptide representing CII245–270 (8) and its analogue peptides containing specific amino acid substitutions were chemically synthesized by a solid phase procedure described previously (10) using an Applied Biosystem (model 430) Peptide Synthesizer (Foster City, CA).

Animals

DBA/1 mice obtained from The Jackson Laboratory (Bar Harbor, ME) were maintained in groups of six in polycarbonate cages and fed standard rodent chow (Ralston-Purina, St. Louis, MO.) and water ad libitum. The environment was specific pathogen free, and sentinel mice were tested routinely for mouse hepatitis and Sendai viruses.

Immunization

For induction of arthritis, mice were immunized with CII at 8 to 12 wk of age as described previously (5). CII was dissolved in 0.01 N acetic acid and emulsified with an equal volume of CFA (5). The resulting emulsion was injected intradermally into the base of the tail. Each mouse received a total volume of 50 µl containing 100 µg of Mycobacterium tuberculosis and 100 µg of Ag.

Treatment with A9 analog peptide

DBA/1 mice were treated with A9 using several different routes of administration. Treatment protocols used in the present study are as follows: 1) each mouse was given one dose of 120 µg of A9 emulsified with IFA i.p. either 2 or 4 wk after CII immunization; 2) each mouse was given by oral gavage eight doses of 100 µg of A9 beginning on day 0 (the day of CII immunization) and continuing every other day until day 17; 3) each mouse was given by intranasal instillation eight doses of 100 µg of A9 beginning on day 0 and continuing every other day until day 17; and 4) each mouse was administered i.v. 300 µg of A9/dose for a total of four doses on days 17, 19, 21, and 24 following CII immunization.

The incidence and severity of arthritis shown represent data taken 6 wk after CII immunization when the control animals reached their peak incidence.

Measurement of serum Ab titers

Mice were bled at 4 wk after immunization to test for Abs reactive with native CII using a modification of an ELISA previously described (11).

Statistical analysis

The incidence of arthritis in various groups of mice was compared using Fisher’s exact test. Ab levels were compared using Student’s t test.

Antibodies

A monoclonal anti-phosphotyrosine Ab PY20 was purchased from Transduction Laboratory (Lexington, KY). A polyclonal Ab recognizing TCR{zeta} peptide was raised in rabbits using a short synthetic peptide (DTYDALHMQTLAPR) corresponding to amino acid residues 151 to 164 of the murine TCR{zeta} chain sequence. A cysteine residue was added to the amino-terminus of the peptide for coupling to maleimide-activated keyhole limpet hemocyanin. The procedures for immunization of rabbits have been described previously (12). The specificity of the Ab we generated has been confirmed using a specific antiserum to TCR{zeta} provided by Dr. Jeffrey V. Ravetch, Laboratory of Biochemical Genetics, Memorial Sloan-Kettering Cancer Center, as described in detail previously (13).

TCR{zeta} chain phosphorylation studies

Preparation of cells. Establishment and characterization of an APC line (M12Aq), {alpha}1(II)-CB11-reactive T cell hybridomas (qcII85.33, 4qcII40), and a CII245–270-reactive T cell line (DBA/1{alpha}A2) have been described previously (8). To induce tyrosine phosphorylation of TCR{zeta} in T cell hybridomas and the T cell line, the cells were treated as follows. APC M12Aq cells (6 x 106) were incubated with or without CII synthetic oligopeptides (300 µg/ml) in RPMI 1640/10% FBS at 37°C for 12 h. The cells were then washed, resuspended in 0.5 ml of RPMI 1640/10% FBS medium, and incubated with either T cell hybridomas or T cell lines (1.2 x 107) in 0.5 ml of RPMI 1640 supplemented with 10% FBS in Eppendorf tubes. The cell mixture was spun at a low speed (500 x g) for 30 s, then incubated at 37°C for 5 min. Stimulation was terminated by adding 1 ml of lysis buffer (20 mM Tris-HCl (pH 7.4) containing 1% Nonidet P-40, 150 mM NaCl, 10% glycerol, 50 mM NaF, 0.2 µM Na3VO4, 1 mM PMSF, 10 µg of leupeptin/ml, and 10 µg of aprotinin/ml). Insoluble materials were removed by centrifugation at 10,000 x g at 4°C for 15 min.

Immunoprecipitation and Western blot. For immunoprecipitation, the clarified cell lysates were mixed with 3 µl of TCR{zeta} antiserum and incubated on ice for 2 h (13). Then, protein A-Sepharose BL-4 (Pharmacia, Piscataway, NJ) was added, and the samples were rotated at 4°C for 30 min. Immunoprecipitates were extensively washed with lysis buffer before suspension in Laemmli’s sample buffer. Proteins were separated on a SDS-12.5% PAGE gel and electrotransferred onto nitrocellulose membranes. After transfer, the membrane was dried at room temperature and washed twice in TBS-T buffer (20 mM Tris-HCl (pH 7.6)/150 mM NaCl and 0.1% Tween 20). The Western blot analysis described previously was used with minor modification (14). Briefly, for detection of phosphorylation of TCR{zeta}, the nitrocellulose membrane was blocked in TBS containing 5% BSA for 2 h, incubated with a monoclonal anti-phosphotyrosine Ab PY20 (1 µg/ml, Transduction Laboratory) in TBS-T/5% BSA for 2 h, and washed four times with TBS-T. The membrane was then incubated with a sheep anti-mouse peroxidase-conjugated Ab (Amersham, Arlington Heights, IL) for 1 h and subjected to enhanced chemiluminescence detection (ECL Western blot kit, Amersham) according to the manufacturer’s protocol. For detection of TCR{zeta}, the membranes were blocked in TBS containing 5% nonfat milk for 2 h, incubated with polyclonal rabbit Abs against TCR{zeta} (affinity purified; 1 µg/ml), followed by incubation with a sheep anti-rabbit peroxidase-conjugated Ab (Amersham).

Measurement of T cell cytokines, IFN-{gamma}, IL-4, and IL-10

Quantitative measurement of murine IFN-{gamma}, IL-4, and IL-10, was performed using a solid phase ELISA based on the sandwich principle. Kits commercially available were used (IFN-{gamma}: Life Technologies, Gaithersburg, MD; IL-4 and IL-10: Endogen, Boston, MA). Briefly, spleens and lymph nodes from DBA/1 mice immunized with CII or A9 emulsified with CFA 10 to 14 days previously were individually minced into single cell suspensions in HBSS and washed three times with HBSS. Pooled splenocytes and lymph node cells were then adjusted to a concentration of 5 x 106 cells/ml and cultured with 100 µg/ml of Ag (synthetic peptides, collagen, or PPD) in DMEM (Life Technologies, Grand Island, NY) supplemented with 5% FBS (HyClone, Logan UT). Supernatants were collected from 72 to 120 h later and either used fresh or frozen at -70°C. Supernatant samples were incubated in microtiter wells coated with an mAb recognizing murine IFN-{gamma}, IL-4, or IL-10. Samples were washed, incubated with a preformed detector complex consisting of a biotinylated second mAb to the appropriate cytokine and an antibiotin-alkaline phosphatase conjugate. The absorbance was measured at 405 nm with a spectrophotometer. A standard curve was obtained by plotting the absorbance vs the corresponding concentration of the standards. Values are expressed as picograms per milliliter. Each sample was tested with duplicate wells.

Class II peptide binding experiments using purified I-A molecules

M12.C3 cells were transfected with A{alpha}q and Aßq cDNA together with a neomycin resistance gene using electroporation to develop I-Aq-expressing cells for these studies (15). For purification of I-Aq, cells were lysed in 2% Nonidet P-40 in PBS, and the lysate was recovered by centrifugation and stored frozen at -70°C. Upon thawing, the lysates were again centrifuged, and filtered through 0.8- and 0.45-µm filters before application to the affinity column. Solubilized I-Aq was purified by passage of the lysate over a protein G orientation column (Pierce, Rockford, IL) coupled with the anti-I-A mAb, M5/114.5.2. The column was then washed with 0.1% SDS and 0.5% Nonidet P-40 in PBS, and the detergent was exchanged with 1% octyl glucoside and 150 mM NaCl at pH 11. The eluate was neutralized immediately with 2 M glycine, pH 2.5. Fractions were analyzed by SDS-PAGE for he presence of I-Aq and concentrated to 1 to 2 mg/ml using an Amicon Stirred Cell (Beverly, MA). The I-Aq was quantitated using a detergent-compatible protein assay (DC Protein Assay, Bio-Rad, Melville, NY), and the quality of the preparation was analyzed by SDS-PAGE.

I-A peptide binding assays were performed by incubating purified I-Aq with 125I-labeled CII (Y257–270) and a mixture of protease inhibitors (16, 17, 18). Five micrograms of purified I-Aq was incubated with approximately 25 pg of labeled peptide in the presence of 1 mM PMSF, 1.3 mM phenanthroline, 6 mM N-ethylmaleimide, 8 mM EDTA, 73 µM pepstatin A, and 135 µM L-p-tosylamino-2-phenylethyl chloromethyl ketone in 1% octyl glucoside-PBS in a total volume of 15 µl. Synthetic peptide, CII257–270, containing a residue of tyrosine at the amino terminus was radioactively labeled by incubation of the peptide with 125I in the presence of chloramine-T. For competitive binding experiments, various quantities of unlabeled peptides were added to the binding assays. After 48 to 72 h at room temperature, the I-A-125I-labeled peptide complexes were separated from free 125I peptides using spin columns containing Sephadex G-50, and the 125I in both the unretarded fractions (I-A bound peptide) and the retarded fractions (unbound peptide) was quantified using a gamma counter. Data are expressed either as the percentage of offered peptide bound to I-Aq or, in the competitive binding experiments, as the percentage of 125I labeled peptide bound.

Passive transfer of cells

Cells for passive transfer studies were obtained from spleens of DBA/1 mice immunized with either A9 or OVA emulsified with CFA 8 days previously. The spleen cells were individually minced into single cell suspensions in HBSS. Erythrocytes were lysed with NH4Cl, and the cells were washed three times in PBS, adjusted to the appropriate concentrations, and injected into naive recipient mice retro-orbitally. Mice were immunized with CII the day after the cell transfer and were observed for arthritis as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The A9 analog suppresses arthritis when administered after the induction of arthritis

We have previously reported that coadministration of the A9 analogue peptide at the time of immunization with CII led to a profound suppression of CIA. However, it remained unknown how effective the analogue peptide might be if administered after immunization with CII or after arthritis was established. To investigate this, A9 was administered i.p. (in IFA) to groups of DBA/1 mice either 2 wk (Fig. 1GoA) after CII immunization (which is just before the onset of arthritis) or 4 wk (Fig. 1GoB) after CII immunization (which is after the development of swollen joints). As shown, A9 peptide was able to significantly alter even established disease when given as a single dose i.p. (p <= 0.02, using Fisher’s exact test). Moreover, the analogue peptide was equally effective when given by other routes: orally, intranasally, or i.v. When the oral and intranasal routes of administration were used, the peptide was administered for a total of eight doses every other day beginning on the day of CII immunization and continuing until day 17 postimmunization. In the case of i.v. administration, each mouse was given four doses of peptide beginning on day 17 after immunization (at the onset of arthritis) and continuing until day 25 postimmunization. A9 administered by the oral, nasal or i.v. routes effectively suppressed the development of new arthritic joints (Table IGo). In each case, the levels of Ab to CII were significantly lower than those in control mice administered OVA (Table IGo).



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  		FIGURE 1. Groups of 24 DBA/1 mice were immunized intradermally in the tail with 100 µg of CII in CFA and then given a single dose of 120 µg of either A9 or OVA i.p. in IFA 2 wk later (A) or 4 wk later (B). Control animals were given OVA. In A, the mice given A9 were significantly different from controls by 35 days after immunization; 4 of 24 A9-treated mice were arthritic compared with 12 of 24 controls (p = 0.02, by Fisher’s exact test). In B, the mice given A9 were also significantly different from controls. On day 45 after immunization, 6 of 24 mice were arthritic compared with 16 of 24 controls (p = 0.004, by Fisher’s exact test).

 

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  		Table I. Effect of route of administration on the suppressive activity of analog A91

 
Phosphorylation of TCR{zeta} in T hybridomas and T cell line in response to CII245–270 and analog peptides

The T cell signaling events induced by A9 peptides were analyzed by comparing the phosphorylation patterns of the TCR{zeta} chain upon TCR engagement of either the analogue peptide A9 or the immunodominant determinant CII245–270. The TCR{zeta} chain is critical for signaling through the TCR, and tyrosine phosphorylation patterns of TCR{zeta} might vary in response to analogue peptides. A T cell line and two T hybridomas that specifically respond to CII245–270 were cultured with APCs previously pulsed with CII peptides, and the TCR{zeta} protein was immunoprecipitated from the cell lysates. Western blot analysis was performed using an anti-phosphotyrosine Ab to analyze the phosphorylation of TCR{zeta}. As shown in Figure 2Go, B, C, and D, similar patterns of TCR{zeta} phosphorylation were observed in a T cell line and two T hybridomas in response to CII peptides. Exposure of the cells to APC previously charged with wild-type peptide (A2) resulted in the appearance of tyrosine-phosphorylated TCR{zeta} of 18 and 21 kDa, representing two phosphorylated isoforms of the TCR{zeta} chain. No phosphorylation was observed in the absence of peptide or in the presence of OVA. Moreover, phosphorylation of TCR{zeta} could not be detected in the immunoprecipitates from the cell lysates exposed to analogue peptides (A9 and A10). Equal amounts of protein were detected when the same membranes were reprobed with Ab to TCR{zeta}, indicating that alteration of the tyrosine phosphorylation pattern of TCR{zeta} was not due to changes in the level of protein. These data suggest that A9 peptide does not induce detectable TCR{zeta} phosphorylation in selected hybridomas and T cell lines.



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  		FIGURE 2. Phosphorylation of TCR{zeta} chain in T cells and T hybridomas. Amino acid sequences of wild-type peptide CII245–270 (A2) and analogue peptides (A9 and A10) are shown in A. B represents hydroxyproline. A dash represents identity at that position with wild-type peptide. The numbers represent the residue positions within type II collagen. The cells from a T cell line (B) and two T hybridomas (C and D) were treated with chick OVA, A2, A9, A10, or buffer control (No Ag) as shown, and Western blotting was performed with anti-phosphotyrosine Ab (upper panel) or antiserum to TCR{zeta} (lower panel).

 
A9 analog peptide did not alter the TCR signaling of T cells when cultured together with wild-type peptide

It is also possible that exposure of T cells to A9 analogue peptide might interfere with signal transduction through TCR-{zeta} induced by wild-type A2. To explore this possibility, we first cultured APCs with wide-type peptide A2 alone or with A2 mixed with analogue A9 or A10 at different ratios. The prepulsed APC then were washed and then exposed to T hybridomas. As shown in Figure 3GoA, neither A9 nor A10 induced phosphorylation of TCR{zeta} at a dose 20-fold higher than that used for A2. Moreover, a ratio of A9 to A2 at as high as 100:1 failed to prevent the induction of phosphorylation of TCR{zeta} chain by wide-type peptide (Fig. 3GoB). These data suggest that neither analogue altered the signaling of wild-type peptide A2 through the TCR. The resulting phosphorylation of the TCR{zeta} when analogues A9 and A10 were presented together with wild-type A2 on APCs did not differ from that with presentation of wild-type peptide alone.



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  		FIGURE 3. The effect of substitutions of CII245–270 peptide on phosphorylation of TCR{zeta} chain in T cell hybridomas. A, APCs were pulsed with A2, A9, A10, A2 plus A9 (A2: A9 = 1:20), or A2 plus A10 (A2: A10 = 1:20) and were used to stimulate T hybridomas. TCR{zeta} proteins were immunoprecipitated with antiserum to TCR{zeta} and analyzed by anti-phosphotyrosine Ab (upper panel) or anti-TCR{zeta} Ab (lower panel). B, APCs were treated with A2 and A9 at various ratios, as indicated, and were used to stimulate T hybridomas. TCR{zeta} proteins were immunoprecipitated with antiserum to TCR{zeta} and were analyzed by anti-phosphotyrosine Ab (upper panel) or Ab to TCR{zeta} (lower panel).

 
Effects of A9 on cytokine production by T cells

Another possible mechanism of action for A9 might be the induction of a suppressive cytokine profile. A9 analogue contains site-directed substitutions at three different positions. Two of the positions, 260 and 263, have been previously identified to be anchoring residues for binding to the I-Aq. The other residue, 261, was demonstrated to be important for interaction with the TCR (7, 15). Therefore, we examined the secretion of three different cytokines, IFN-{gamma} (Th1), IL-10 (Th2), and IL-4 (Th2), when pooled splenocytes and draining lymph node cells from CII-immunized mice were cultured in vitro with various analogue peptides (Table IIGo). As expected, the cytokine response to wild-type A2 peptide was predominantly a Th1 response, with 7875 pg/ml IFN-{gamma}, 3132 pg/ml IL-10, and 45 pg/ml IL-4. Analog peptides with substitutions at the anchoring residues, with individual substitutions at 260 (B4), 263 (B3), or both (A4), gave negligible cytokine responses, either Th1 or Th2. The analogue peptide that contained a substitution at the TCR contact site 261 (A6) gave both a Th1 and a Th2 response, but decreased compared with that obtained with the wild-type peptide. Interestingly, the analogue peptide A9, which contained substitutions at 260, 261, and 263 had a secretion profile that was entirely Th2. Cells responding to A9 secreted 2680 pg/ml of IL-10 and 60 pg/ml of IL-4. Two other peptides, A10 and N (CII362–386), gave decreased cytokine responses compared with that of wild-type peptide, while PPD induced a strong Th1 response and a negligible Th2 response (Table IIGo).


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  		Table II. Immune response to peptides after immunization with CII1

 
Effects on binding to I-Aq

Another possible explanation for the effectiveness of A9 in down-regulating arthritis might be that it binds to the MHC with higher affinity than the wild-type peptide, causing inhibition of T cell activation by blocking the Ag binding site of MHC molecules, resulting in effective competitive inhibition of A2. To evaluate this possibility, we set up a soluble I-Aq-peptide binding assay. Various concentrations of A9 analogue peptide and various control peptides were tested for the ability to compete with labeled wild-type 245–270 peptide, establishing a relative comparison of the binding affinities of each peptide for I-Aq. As expected, the wild-type peptide competed effectively with the labeled wild-type for binding to I-Aq, as well as two other control peptides, HEL16–30 and A10. On the other had, A9 analogue did not compete well with the labeled wild-type peptide (Fig. 4Go), even at a concentration ratio of 1/1000, suggesting that the A9 analogue does not bind well to I-Aq. In a second set of experiments, A9 peptide was directly labeled and tested for binding to I-Aq (data not shown) and again showed poor ability to bind to the MHC. The fact that A9 peptide binds less efficiently to I-Aq than wild-type peptide makes competition for binding at the level of the I-Aq molecule an unlikely explanation for the effectiveness of A9 in suppression of CIA.



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  		FIGURE 4. Comparisons of relative binding affinities of peptides to soluble I-Aq class II molecules. A competitive peptide binding assay using soluble I-Aq was performed to compare the relative binding affinity of the A9 peptide to those of other peptides known to bind to this class II molecule. 125I-labeled CII (Y257–270) was used as the indicator peptide, and various concentrations of competitor peptides were added to each assay. Data are expressed as the percentage of 125I-labeled CII (Y257–270) bound in the absence of competitor and are representative of at least two experiments. The percentage of 125I-labeled CII (Y257–270) bound to I-Aq in the absence of competitor ranged from 3.6 to 5.5.

 
Immunization with A9

Even though A9 binds poorly to MHC, CII-immune T cells (possibly a subset) respond to A9 with a significant Th2-type cytokine profile. In a second set of experiments, DBA/1 mice were immunized with A9 analogue peptide emulsified with CFA, and the resulting splenocytes and lymph node cells were cultured with various Ags. Again, the responses to wild-type peptide and {alpha}1(II) remained predominantly Th2, while the response to PPD was Th1 (Table IIIGo). This can be compared with control mice immunized with wild-type A2 peptide, which generated predominantly a Th1 response to A2 and {alpha}1(II).


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  		Table III. Immunization with either wild-type A2 or analog peptide A91

 
Taken together, these data suggest that a population of cells can be induced to respond to the A9 peptide with a predominantly Th2 phenotype. The ability to induce the secretion of suppressive cytokines may explain the profound suppressive effect A9 has on CIA.

Transfer of suppression of arthritis with A9-immune splenocytes

To confirm the importance of an active secretion of Th2 cytokines on the modulation of CIA, cell transfer experiments were performed. DBA/1 mice were immunized with A9 emulsified with CFA, and splenocytes were transferred i.v. to naive mice. The recipient mice were then immunized with CII and observed for arthritis (Table IVGo). As predicted, the A9-immune splenocytes caused a decrease in the incidence of arthritis with a final incidence of 30% compared with a 90% incidence in control mice given splenocytes from mice immunized with OVA. The total Ab response was also significantly reduced (30 ± 12 from mice given A9-immune cells compared with 62 ± 14 from mice given OVA-immune cells; p <= 0.05). Taken together, these data suggest that the suppression of CIA induced by the A9 analogue peptide is due to its ability to induce a suppressive, Th2-like cytokine response, and this suppression can be actively transferred by A9-immune splenocytes.


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  		Table IV. Transfer of suppression of arthritis with splenocytes1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The identification of analogue peptides that affect the induction and severity of disease in animal models of autoimmunity (19, 20) has raised considerable interest in the use of altered peptide ligands as Ag-specific therapeutic agents. If one can identify the self-antigenic determinant responsible for a T cell-mediated autoimmune disease, for example, then the generation and use of analogues could prevent or inhibit the disease process. In support of this, we have recently reported that an analogue peptide of the immunodominant determinant located within {alpha}1(II)-CB11 prevented the induction of CIA when administered to mice at the time of immunization with CII (8).

In the present paper we now demonstrate that A9 is equally effective even when the peptide is administered after arthritis has been induced. Moreover, the down-regulation of established CIA occurs by shifting the CII-specific T cell response toward a Th2-type profile. Th2 cytokines, such as IL-4, IL-10, and TGF-ß, effectively down-regulate inflammatory responses in mice (21, 22). An in vivo role of Th2 cytokines in CIA was suggested by Mauri et al., who found IL-10 and low, but persistent, levels of IL-4 in lymph nodes late in disease when CIA was in remission (23). Moreover, mice given IL-4 either parenterally or as IL-4 gene-transfected CHO cells developed CIA at a lower incidence than controls (24, 25), and daily i.p. injections of murine rIL-10 caused mice to develop a milder CIA (26). It thus appears that Th2-type cytokines attenuate the inflammation of CIA, while Th1-type cytokines support inflammation (27, 28).

A9 is unique in requiring three critical substitutions to be effective in suppressing CIA. The important residues are of two types: one anchors the peptide to the MHC molecule, and the other interacts with the TCR. Other investigators have reported that peptides of low MHC binding affinity may favor the generation of Th2 responses (29). When mice were immunized with the immunodominant peptide of collagen type IV, which binds with high affinity to I-As and with low affinity to I-Ab, Th1-like cells were selectively induced in I-As -bearing mice, and Th2-like cells were induced in I-Ab mice. Analog peptides of collagen type IV with decreased binding affinity to I-As stimulated T cells to produce primarily IL-4, rather than IFN-{gamma} (29). In the CIA animal model we have demonstrated that A9 binds less well to I-Aq than does wild-type A2, but alteration of the two residues binding I-Aq is not enough to induce Th2 cytokines. An additional third substitution is required.

When TCR contact residues of a cytochrome c peptide were systematically altered, CD4+ T cells specific for cytochrome c could also be manipulated to produce predominantly IL-4 (30). A panel of analogues of a determinant of cytochrome c varying only at the TCR contact sites could prime T cells to secrete IL-4, while immunization with wild-type peptide led to a predominance of IFN-{gamma} secretion (30). Using the animal model of experimental allergic encephalitis (EAE), Das and co-workers preimmunized animals with an analogue peptide of myelin proteolipid protein PLP139–141, which caused a protective Th2 response rather than the encephalitogenic Th1 cytokine profile (31). This analogue contains a single alanine substitution at a TCR contact residue at position 144, which is adjacent to an MHC contact residue at position 145. Nevertheless, alteration of a single TCR contact residue does not always induce a Th2 response. Substitution of residue 261 of the wild-type A2 merely decreased the magnitude of the overall cytokine response, both Th1 and Th2. In a similar vein, Kersh and co-workers demonstrated that an analogue (32) could retain biologic function only if the changes in size, hydrophobicity, or conformation of the TCR contact residue were small. Taken together, these data suggest that alteration of either MHC binding affinity or the TCR contact residue may favor the generation of Th2 responses. In the case of A9, three residues, two MHC contact and one TCR contact, required alteration to induce Th2 cytokines and suppress arthritis.

A9 differs from certain analogues that inhibit specific T cell responses by competition for binding to the MHC molecule (33, 34). Peptides that bind with high affinity to the MHC have successfully prevented murine EAE (35, 36). A9 also differs from TCR antagonists, which occupy the TCR and prevent the induction of normal signals (37, 38, 39). Using two T cell hybrids and an Ag-specific T cell line, we were unable to demonstrate that A9 altered TCR{zeta} chain phosphorylation produced in response to the wild-type A2. Recent data have described a distinct pattern of {zeta}-chain phosphorylation accompanied by the failure to activate ZAP-70 kinase following TCR ligation (40, 41). We have been unable to demonstrate any phosphorylation of the TCR{zeta} in response to the A9 peptide, and therefore believe it unlikely that our peptide is a partial agonist.

Das and co-workers conclude that T cell clones reactive with the A144 analogue of PLP that suppress EAE, are not the same T cell clones and do not have the same TCR structures as the predominantly Th1 clones that recognize PLP139–141 and are encephalitogenic (31). Disease-inducing clones do not tolerate a substitution at W144. Similarly, the CII-reactive hybridomas and the T cell line described in Figures 2Go and 3Go of this manuscript had no detectable response to A9. These data suggest that these are not the same T cell clones that A9 can induce to secrete Th2 cytokines and suppress CIA. We conclude that A9 probably primes a second set of T cell population capable of cross-reacting with CII260–270 with a primarily Th2 response.

There are strong theoretical and practical reasons to suggest that an immunologically specific therapy of autoimmune diseases is preferable to the use of immunologically nonspecific drugs and Abs. Analog peptides of CII, a cartilage-specific protein, which can be designed to down-regulate established arthritis, could have enormous potential as specific immunotherapeutic agents for autoimmune arthritis.


   Acknowledgments
 
We thank Mike Wardlow and Karen Whittington for excellent technical support.


   Footnotes
 
1 This work was supported in part by U.S. Public Health Service Grants AR-39166 and AR-43589, program-directed funds from the Veteran’s Administration, and funds from the Arthritis Foundation. Back

2 Address correspondence and reprint requests to Dr. Linda K. Myers, 956 Court Ave., Room G326, Memphis, TN 38163. Back

3 Abbreviations used in this paper: CIA, collagen-induced arthritis; CII, type II collagen; CB, cyanogen bromide; TBS-T buffer, 20 mM Tris-HCl (pH 7.6)/150 mM NaCl and 0.1% Tween 20; PPD, purified protein derivative; EAE, experimental allergic encephalitis; PLP, proteolipid protein. Back

Received for publication December 16, 1997. Accepted for publication June 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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