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The Journal of Immunology, 1998, 160: 1132-1138.
Copyright © 1998 by The American Association of Immunologists

Alloantigen-Reactive Th1 Development in IL-12-Deficient Mice1

Joseph R. Piccotti2,*, Kewang Li*, Sherri Y. Chan*, Jessica Ferrante{ddagger}, Jeanne Magram{ddagger}, Ernst J. Eichwald§ and D. Keith Bishop*,{dagger}

* Department of Surgery, Section of General Surgery, and {dagger} Department of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, MI 48109; {ddagger} Department of Inflammation and Autoimmune Diseases, Hoffmann-La Roche Inc., Nutley, NJ 07110; and § Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-12p70, a 70- to 75-kDa heterodimer consisting of disulfide-bonded 35-kDa (p35) and 40-kDa (p40) subunits, enhances Th1 development primarily by its ability to induce IFN-{gamma} production by NK and Th1 cells. Although homodimers of the p40 subunit of IL-12 are potent IL-12 receptor antagonists in some systems, we have reported that p40 homodimer may accentuate alloreactive CD8+ Th1 function. To test the role of endogenously produced p40 in alloimmunity, Th1 development was assessed in either IL-12 p35 knockout (p35-/-) mice, the cells of which are capable of secreting p40, or p40 knockout (p40-/-) mice. Compared with IL-12 wild-type controls, splenocytes obtained from both p35-/- and p40-/- mice produced markedly less IFN-{gamma} after in vitro stimulation with Con A or alloantigens. Interestingly, in vivo-sensitized Th1 were detected in both p35-/- and p40-/- cardiac allograft recipients. However, in vivo Th1 development was enhanced in p35-/- recipients compared with p40-/- animals, suggesting that endogenous p40 produced in p35-/- mice may stimulate alloreactive Th1. Indeed, neutralizing endogenous p40 with anti-IL-12 p40 mAb reduced Th1 development in p35-/- allograft recipients to that seen in p40-/- mice. To determine whether Th1 development that occurred in the absence of IL-12p70 and p40 required IFN-{gamma}, p40-/- allograft recipients were treated with anti-IFN-{gamma} mAb. Neutralizing IFN-{gamma} did not inhibit in vivo Th1 development in p40-/- recipients and resulted in a unique pathology of rejection characterized by vascular thromboses. Collectively, these data suggest that 1) endogenous p40 may substitute for IL-12p70 in alloantigen-specific Th1 sensitization in vivo and 2) in vivo alloreactive Th1 development may occur independent of IL-12 and IFN-{gamma}, suggesting an alternate Th1-sensitizing pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-12 is a key cytokine involved in the regulation of Th1/Th2 balance in both in vitro and in vivo immune responses (reviewed in Refs. 1 and 2). IL-12 indirectly promotes Th1 and inhibits Th2 development by inducing IFN-{gamma} production by NK and Th1 cells (3, 4, 5, 6, 7, 8). In addition, IL-12 has direct stimulatory and inhibitory activities on Th1 and Th2, respectively (9, 10, 11, 12). Th1 initiates allograft rejection by promoting both CTL and delayed-type hypersensitivity (DTH)3 responses, which serve as the terminal effector mechanisms of allograft rejection (13, 14). Since Th2 are antagonistic to many of the activities of Th1 (15, 16), it has been proposed that preferential induction of alloantigen-reactive Th2 may inhibit Th1-mediated rejection responses, thereby promoting allograft tolerance (17, 18). A likely target for such an inductive therapy is IL-12, since IL-12 antagonism inhibits Th1- and promotes Th2-driven immune responses in several experimental models (19, 20, 21, 22). However, we have reported that IL-12 antagonism does not inhibit in vivo sensitization of IFN-{gamma}-producing cells (23), suggesting that IL-12 is not requisite for alloreactive Th1 development.

IL-12 is a 70- to 75-kDa heterodimer (IL-12p70) consisting of disulfide-bonded 35-kDa (p35) and 40-kDa (p40) subunits (24, 25). The biologic activities of IL-12p70 are mediated through the high affinity IL-12R, which is composed of IL-12Rß1 and IL-12Rß2 chains (26). It has been suggested that the biologic activity of heterodimeric IL-12p70 requires the interaction of p40 with IL-12Rß1 and the interaction of p35 with IL-12Rß2 (26, 27). This interaction of IL-12p70 with its high affinity receptor induces phosphorylation of the Janus kinases JAK2 and TYK2 (28) and of STAT3 and STAT4 (29). Recent evidence suggests that IL-12Rß1 interacts with TYK2, while IL-12Rß2 interacts with JAK2 (30). The importance of IL-12Rß2 in IL-12p70 signaling has been emphasized by two recent reports which document that loss of IL-12 responsiveness by Th2 is related to down-regulation of IL-12Rß2 expression (31, 32). These studies (31, 32) further document a requirement for IL-12Rß2 expression in IL-12p70-induced STAT4 phosphorylation. However, the role of IL-12Rß1 in IL-12-mediated signal transduction is less well defined. Recent studies have shown that IL-12Rß1 is necessary for IL-12p70 signaling (33). Further, homodimers of p40, which bind IL-12Rß1, are potent competitive inhibitors of IL-12p70 in some experimental systems (34, 35). Since secretion of IL-12p70 is associated with excess production of the p40 subunit (25, 36), it has been proposed that excess production of p40 may down-regulate IL-12-mediated immune responses (1, 37). However, we have recently reported that the antagonistic properties of p40 homodimer preferentially target alloreactive CD4+ Th1 and that p40 homodimer enhances, rather than inhibits, alloantigen-specific CD8+ Th1 development (38). It is possible that this differential effect of p40 homodimer on CD4+ vs CD8+ T cells reflects altered signaling via IL-12Rß1 on these T cell subsets.

In the present study, IL-12 p35 knockout (p35-/-) and p40 knockout (p40-/-) mice were used to evaluate the effects of endogenously produced p40 on alloreactive Th1 development. Mutation of the p40 gene in p40-/- mice inhibits the expression of the p40 subunit (39), thereby ablating production of bioactive IL-12p70. The p35 subunit, which may be produced by p40-/- mice, is not secreted in the absence of p40 (36). Similarly, p35-/- mice fail to produce IL-12p70. However, p35-/- mice secrete normal concentrations of the p40 subunit compared with IL-12 wild-type (IL-12 WT) mice (40). Hence, this system allowed us to explore the requirement for biologically active IL-12 in alloreactive Th1 development and to define the role of endogenously produced p40 in alloimmune responses in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female C57BL/6 and BALB/c mice between 6 and 12 wk of age were obtained from Charles River Laboratories (Raleigh, NC). Generation of p35-/- and p40-/- mice of both C57BL/6 and BALB/c backgrounds have been described previously (39, 40). These mice were generated on the 129/Sv/Ev background, backcrossed to C57BL/6 or BALB/c mice for five generations, and then intercrossed to generate homozygotes.

Medium

The culture medium used in these studies was DMEM supplemented with 1.6 mM L-glutamine, 0.27 mM L-asparagine, 10 mM HEPES buffer, 1.0 mM sodium pyruvate, 100 U/ml penicillin/streptomycin, 2% FCS (all obtained from Life Technologies, Grand Island, NY), and 5 x 10-5 M 2-ME (Sigma Chemical Co., St. Louis MO).

In vitro Th1 development

Spleens obtained from naive IL-12 WT, p35-/-, or p40-/- C57BL/6 mice were processed into single-cell suspensions by gently passing tissues through wire mesh. Cells were then washed and resuspended in supplemented tissue culture medium. To investigate the role of IL-12 in mitogen-stimulated Th1/Th2 cytokine production, splenocytes (2 x 106 cells/ml) were incubated for 72 h with 1 µg/ml Con A (Sigma Chemical Co.). To inhibit IL-12 activity, 10 µg/ml of neutralizing polyclonal goat anti-IL-12 Abs were added (provided by Dr. Maurice Gately, Hoffmann-La Roche Inc., Nutley, NJ). Where indicated, cultures were supplemented with 1 ng/ml murine rIL-12 (also provided by Dr. Gately) to assess the effect of exogenous IL-12 on Con A-stimulated cytokine production by splenocytes of IL-12-deficient mice. Culture supernatants were harvested at 72 h, and cytokine concentrations were measured by ELISA.

To assess alloantigen-specific Th1 development, splenocytes (1 x 106 cells/ml) isolated from naive IL-12 WT, p35-/-, or p40-/- C57BL/6 mice were incubated for 5 days with irradiated (5000 rads) IL-12 WT, p35-/-, or p40-/- BALB/c splenocytes (1 x 106 cells/ml), respectively. To determine whether splenocytes from IL-12-deficient mice were receptive to IL-12 stimulation, 1 ng/ml of murine rIL-12 was added to primary MLC. Resulting cell populations (1 x 106 cells/ml) were harvested, washed three times, and restimulated with the appropriate irradiated BALB/c stimulator cells (1 x 106 cells/ml). MLC supernatants were collected after 24 h (IL-4 and IL-10) or 72 h (IFN-{gamma}), and cytokine concentrations were measured by ELISA.

In vivo Th1 development in cardiac allograft recipients

Intact BALB/c-H-2d hearts were anastomosed to the great vessels in the abdomens of C57BL/6-H-2b mice as described by Corry et al. (41). Where indicated, p35-/- or p40-/- mice were used as both allograft donors and recipients. In this model, the transplanted heart is perfused with the recipient’s blood and resumes contractions until acutely rejected, which occurs in unmodified WT recipients of this strain combination in ~8 to 9 days (38). Graft function was evaluated by daily abdominal palpation. Myocyte damage and intensity of graft-infiltrating cells were assessed by routine hematoxylin and eosin (H & E)-stained paraffin-embedded fragments of transplanted allografts. To monitor in vivo Th1 development, splenocytes (1 x 106 cells/ml) obtained from allograft recipients were restimulated with the appropriate irradiated BALB/c stimulator cells (1 x 106 cells/ml) for 72 h, and the concentration of IFN-{gamma} was measured by ELISA. This assay detects in vivo-primed Th1, in that splenocytes from naive, nontransplanted mice produce minimal or undetectable concentrations of IFN-{gamma} under these conditions (23, 38).

Two approaches were used to determine the effects of endogenous p40 and IFN-{gamma} on in vivo Th1 development. 1) To neutralize endogenous p40 produced by p35-/- mice (40), allograft recipients received i.p. injections of anti-IL-12 p40 mAb (1 mg) on days 0, 1, and 3 posttransplantation (also provided by Dr. Gately). 2) To neutralize endogenous IFN-{gamma} in p40-/- allograft recipients, animals were injected i.p. with 1 mg anti-IFN-{gamma} mAb (R4-6A2) on days 0, 1, and 3 posttransplantation. At 5 µg/ml, anti-IFN-{gamma} mAb completely neutralizes at least 10 ng/ml IFN-{gamma}, as determined by ELISA (unpublished observation).

Cytokine ELISA

Experimental samples (100 µl) were added in triplicate to plates coated with 5 µg/ml rat anti-mouse IFN-{gamma}, IL-4, or IL-10 capture Abs (PharMingen, San Diego, CA). Standards were set up by preparing twofold dilutions of murine rIFN-{gamma}, IL-4, and IL-10 (PharMingen), with starting concentrations of 25, 2.5, and 10 ng/ml, respectively. Following a 1-h incubation at room temperature, plates were washed three times with 0.05% Tween-20 in PBS. One hundred microliters of rat anti-mouse secondary biotinylated Abs (1 µg/ml) (PharMingen) were then added, and plates were incubated at room temperature for 45 min. Plates were then washed three times with 0.05% Tween-20 in PBS, and 100 µl of avidin-peroxidase (Sigma Chemicals, St. Louis MO) were added. Following a 30-min incubation at room temperature, plates were washed three times with 0.05% Tween-20 in PBS, and 100 µl of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate (Sigma Chemical Co.) were then added to each well. After 20 to 30 min, absorbance was determined at 405 nm by an EL 312e microplate reader (Bio-Tek Instruments, Winooski, VT). Sample cytokine concentrations were calculated from a standard curve. The sensitivity of this assay is ~300 pg/ml for IFN-{gamma}, 100 pg/ml for IL-4, and 150 pg/ml for IL-10.

Depletion of CD4+ and/or CD8+ T cells

Ascites was produced in pristane-treated athymic BALB/c mice bearing the hybridoma GK1.5 as a source of anti-CD4 mAb or hybridoma 2.43 as a source of anti-CD8 mAb. Ab was precipitated with 40% ammonium sulfate, reconstituted to the original volume in PBS, and dialyzed. These mAb preparations maintain their maximal activity in complement-mediated cytolysis assays at dilutions of 1:1000 or greater. For T cell depletion, splenocytes (5 x 106/ml) obtained from C57BL/6 mice bearing BALB/c hearts were incubated with anti-CD4 mAb, anti-CD8 mAb, or both mAbs (diluted 1:500) for 30 min on ice, centrifuged, and washed three times with ice-cold HBSS (Life Technologies, Grand Island, NY). Ab-coated cells were then incubated for 45 min at 4°C with Dynabeads (M-450 sheep anti-rat IgG, DYNAL, Lake Success, NY) with a bead to target cell ratio of 4:1. Targeted cells attached to beads were collected using a magnet, and the resulting cell populations were obtained for functional analyses. Depletion of CD4+ and/or CD8+ cells (<2%) was verified by flow cytometry before each experiment using anti-CD4:FITC or anti-CD8:FITC Ab (PharMingen). To further verify that cells were depleted, rather than coated with the primary mAb, samples were stained with FITC-conjugated goat anti-rat IgG.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Role of IL-12 in mitogen-driven Th1/Th2 cytokine production

To determine the role of IL-12 in Th1/Th2 cytokine production following mitogen stimulation, C57BL/6 splenocytes isolated from control (IL-12 WT) or IL-12-deficient (p35-/- and p40-/-) mice were stimulated with Con A for 72 h, and supernatant cytokine concentrations were determined by ELISA. IL-12 WT splenocytes secreted high concentrations of IFN-{gamma} (21.86 ng/ml) after Con A stimulation (Table IGo). IFN-{gamma} production by splenocytes isolated from both p35-/- and p40-/- mice was readily detectable, although IFN-{gamma} concentrations were markedly lower than in controls (p < 0.001; one-way ANOVA, Scheffé’s test post hoc). A reduced concentration of IFN-{gamma} in IL-12-deficient cultures was not associated with a decrease in the ability of these cells to proliferate in response to Con A, in that proliferative responses of splenocytes obtained from IL-12 WT, p35-/-, and p40-/- mice were equivalent. (data not shown). To determine whether the observed IFN-{gamma} production following mitogen stimulation was dependent on IL-12, cultures were supplemented with anti-IL-12 Abs (10 µg/ml). Neutralizing IL-12 significantly suppressed IFN-{gamma} secretion in IL-12 WT splenocytes stimulated by Con A (21.86 ng/ml vs 4.10 ng/ml), indicating that endogenous IL-12 contributed to high level IFN-{gamma} production by control splenocytes. As expected, neutralizing IL-12 had no effect on IFN-{gamma} production by IL-12-deficient splenocytes. This observation 1) confirms previous reports (39, 40) that the targeted IL-12 alleles are null in the IL-12 knockout mice and 2) indicates that low level IFN-{gamma} production occurs in the absence of IL-12p70. However, IL-12-deficient splenocytes were responsive to IL-12 stimulation, since supplementing cultures with exogenous rIL-12 (1 ng/ml) markedly augmented Con A-stimulated IFN-{gamma} production (Table IGo).


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  		Table I. Mitogen-driven cytokine production by splenocytes obtained from IL-12-deficient mice1

 
Splenocytes isolated from IL-12-deficient mice produced similar concentrations of IL-10 after Con A stimulation when compared with IL-12 WT cells, and the addition of neutralizing anti-IL-12 Abs did not alter IL-10 secretion (Table IGo). Interestingly, the addition of exogenous rIL-12 to cultures did not inhibit IL-10 production but appeared to enhance it. IL-4 (Table IGo) and IL-5 (data not shown) production was not detected after Con A stimulation in any of the experimental groups, indicating that the absence of IL-12 did not lead to an overt dominance of a Th2 response by Con A-stimulated C57BL/6 splenocytes. IL-4 was readily detectable in supernatants of IL-12 WT, p35-/-, and p40-/- BALB/c splenocytes after mitogen stimulation (data not shown). These data are consistent with the observation that BALB/c mice are predisposed to generate Th2 cytokines (42). However, IL-4 production was not markedly enhanced following Con A stimulation of IL-12-deficient BALB/c splenocytes when compared with IL-12 WT splenocytes (data not shown).

In vitro alloreactive Th1 development

To evaluate the role of IL-12 in alloantigen-specific Th1/Th2 development, naive splenocytes obtained from IL-12 WT, p35-/-, or p40-/- mice were incubated for 5 days with allogeneic splenocytes in primary MLC which were left unmodified or supplemented with rIL-12. Resulting cell populations were restimulated with alloantigens, and in vitro Th1/Th2 cytokine production was determined by ELISA. As previously reported (38), primed IL-12 WT splenocytes secreted high concentrations of IFN-{gamma} after restimulation with alloantigens (7.00 ng/ml) (Table IIGo). Splenocytes obtained from p35-/- or p40-/- mice also secreted IFN-{gamma} after restimulation with alloantigens, albeit to a lesser degree than IL-12 WT cells (p < 0.001; one-way ANOVA, Scheffé’s test post hoc). The decrease in alloantigen-stimulated IFN-{gamma} production in IL-12-deficient mice was not associated with a decrease in the ability of the cells to proliferate in response to alloantigens (data not shown). In all three groups, IL-4, IL-10 (Table IIGo), and IL-5 (data not shown) were not detected, indicating that Th1 dominated the in vitro alloantigen-driven responses in either the presence or the absence of IL-12.


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  		Table II. IL-12 and alloantigen-specific Th1 development in vitro1

 
As with Con A stimulation, rIL-12 markedly augmented alloreactive Th1 development in vitro in all experimental groups (Table IIGo). Likewise, rIL-12 enhanced IL-12 WT, p35-/-, and p40-/- splenocyte proliferation in the MLC (data not shown). Finally, the addition of exogenous rIL-12 to cultures stimulated the secretion of low concentrations of IL-10 by alloantigen-stimulated splenocytes, supporting recent reports that IL-12 induces concomitant secretion of IFN-{gamma} and IL-10 (43, 44).

Cardiac allograft rejection in IL-12-deficient mice

Cardiac allograft function was monitored by daily palpation in IL-12 WT, p35-/-, or p40-/- C57BL/6 graft recipients of respective IL-12 WT, p35-/-, or p40-/- BALB/c hearts. As previously reported (38), the mean allograft survival in unmodified IL-12 WT recipients was ~8 days (Table IIIGo). In both p35-/- and p40-/- recipients, cardiac allografts were rejected in an accelerated fashion by day 7 posttransplantation. These results are in keeping with our previous report that rejection occurs in the absence of biologically active IL-12p70 (23).


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  		Table III. Cardiac allograft rejection in IL-12-deficient mice1

 
In vivo sensitization of IFN-{gamma}-producing cells in IL-12-deficient mice

To determine whether cardiac allograft rejection in IL-12-deficient mice was associated with persistent Th1 development in vivo, splenocytes obtained from C57BL/6 allograft recipients were restimulated for 72 h with donor alloantigens, and supernatant concentrations of IFN-{gamma} were measured by ELISA (Fig. 1Go). Interestingly, splenocytes obtained from p35-/- recipients secreted similar concentrations of IFN-{gamma} relative to IL-12 WT mice (p35-/- = 9.48 ng/ml vs IL-12 WT = 10.29 ng/ml). Further, IFN-{gamma} production was readily detectable in p40-/- allograft recipients (3.14 ng/ml), albeit at a lower concentration than observed in IL-12 WT controls or IL-12 p35-/- animals (p < 0.001; one-way ANOVA, Scheffé’s test post hoc). Since p35-/- but not p40-/- mice are capable of producing IL-12 p40 (40) and since p40 homodimer enhances CD8+ alloantigen-reactive Th1 development in vitro (38), these observations suggest that endogenously produced p40 may contribute to Th1 development in p35-/- allograft recipients. To test this possibility, p35-/- cardiac allograft recipients were treated with anti-IL-12 p40 mAb to neutralize endogenous p40. As expected, anti-IL-12 p40 mAb treatment did not alter graft rejection (Table IIIGo). Importantly, neutralizing endogenous p40 reduced in vivo Th1 development in p35-/- allograft-bearing mice to the concentration observed in p40-/- recipients (Fig. 1Go, p < 0.001; Student’s t test). Hence, alloantigen-reactive Th1 developed in the absence of biologically active IL-12p70, and endogenously produced p40 may substitute for IL-12p70 in Th1 sensitization in vivo.



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  		FIGURE 1. In vivo development of alloantigen-specific Th1 in IL-12-deficient mice. IL-12 WT, p35-/-, or p40-/- C57BL/6 mice were transplanted with IL-12 WT, p35-/-, or p40-/- BALB/c hearts, respectively. To assess in vivo Th1 development, splenocytes (1 x 106 cells/ml) obtained from cardiac allograft recipients were restimulated in vitro with donor alloantigens (appropriate irradiated BALB/c splenocytes; 1 x 106 cell/ml). Splenocytes obtained from naive C57BL/6 mice and stimulated with irradiated BALB/c splenocytes served as a negative control for the in vivo sensitization of Th1. Supernatants were collected after 72 h, and the concentration of IFN-{gamma} was measured by ELISA. Results are expressed as the mean concentration of IFN-{gamma} in triplicate samples ± SD. Data are representative of three separate experiments. ND indicates not detectable.

 
Phenotype of alloantigen-reactive Th1 in p35-/-mice

We have previously reported that IL-12 p40 homodimer stimulates alloantigen-reactive CD8+ but not CD4+ Th1 development in vitro. To determine the phenotype of Th1 responsive to endogenously produced p40 in vivo, splenocytes from p35-/- allograft recipients were depleted of CD4+, CD8+, or both CD4+ and CD8+ T cells before addition to the in vitro assay for IFN-{gamma} production (Fig. 2Go). While depletion of CD4+ cells had no effect on IFN-{gamma} production, depletion of CD8+ cells markedly decreased Th1 function. Depletion of both CD4+ and CD8+ cells further reduced IFN-{gamma} secretion by ~98%. These data indicate that alloantigen-reactive Th1 that develop in p35-/- are predominantly CD8+ T cells.



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  		FIGURE 2. Alloantigen-reactive Th1 responding to endogenously produced p40 are CD8+ T cells. Splenocytes were obtained from C57BL/6 p35-/- recipients of BALB/c p35-/- cardiac allografts 7 days after transplantation. Splenocytes were depleted of CD4+, CD8+, or both CD4+ and CD8+ cells by immunomagnetic bead sorting as described in Materials and Methods. Nondepleted and selected populations (1 x 106 cells/ml) were stimulated with irradiated BALB/c p35-/- splenocytes. Supernatants were collected at 72 h, and the concentration of IFN-{gamma} was measured by ELISA. Results are expressed as the mean concentration of IFN-{gamma} in triplicate samples ± SD. Data are representative of three separate experiments.

 
Neutralizing IFN-{gamma} does not prevent in vivo alloantigen-specific Th1 development in p40-/- allograft recipients

Since alloreactive Th1 development occurred in the absence of both IL-12p70 and p40, we asked whether endogenously produced IFN-{gamma} was necessary for Th1 responses in p40-/- mice. To this end, p40-/- allograft recipients were treated with neutralizing anti-IFN-{gamma} mAb (Fig. 1Go). Interestingly, neutralizing IFN-{gamma} did not inhibit Th1 sensitization in p40-/- mice, indicating that alloantigen-specific Th1 development may proceed in the absence of both IL-12 and IFN-{gamma}. Further, allografts were uniformly rejected by day 7 in these mice (Table IIIGo), suggesting that IFN-{gamma}- and IL-12-independent effector mechanisms were operative in the rejection process.

Neutralizing IFN-{gamma} in p40-/- cardiac allograft recipients had a unique effect on the pathology of rejection (Fig. 3GoA). In these animals, graft rejection was characterized by degeneration of the coronary vessel walls, intravascular thrombosis, extensive myocyte necrosis, and a virtual absence of an inflammatory infiltrate. This contrasted the pathology of rejection in untreated p40-/- mice, in which myocytes were relatively well preserved, a heavy mononuclear cell infiltrate was present, and many vessels were occluded by cells rather than thrombi (Fig. 3GoB). Further, this unique type of rejection was not observed in anti-IFN-{gamma}-treated IL-12 WT recipients (data not shown). These observations suggest that alternate (humoral?) effector mechanisms surface in the absence of both IL-12 and IFN-{gamma}.



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  		FIGURE 3. Distinct pathology of rejection in IL-12 p40-/- cardiac allograft recipients following endogenous IFN-{gamma} neutralization. C57BL/6 p40-/- recipients of BALB/c p40-/- cardiac allografts were either left untreated or injected i.p. with anti-IFN-{gamma} mAb on days 0, 1, and 3 following transplantation. On day 7, allografts were harvested for histologic evaluation. A, H & E-stained section of allograft from p40-/- recipient treated with neutralizing anti-IFN-{gamma} mAb (x400). Note thrombosed lumen of vessel (T) and degenerating vessel wall (VW). Also note that the majority of myocytes are necrotic (arrows), despite minimal mononuclear cell infiltration. B, H & E-stained section of graft from unmodified (no treatment) p40-/- cardiac allograft recipient (x400). Note cellular occlusion of vessel lumen and relatively preserved vessel wall (VW). Also note numerous viable myocytes (arrows) as evidenced by the presence of myocyte nuclei and increased mononuclear cell infiltrate relative to A. Results are representative of 8 to 10 individual transplants for each experimental group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current study was initiated to clarify an apparent discrepancy between previous reports that document a requirement for IL-12 in Th1 development in several experimental models (19, 20, 21, 22) and our recent report that IL-12 is not necessary for the development of alloantigen-reactive Th1 (23). Further, we have reported that IL-12p40 homodimer stimulates alloreactive CD8+ Th1 (38), whereas others have found that p40 homodimer functions as an IL-12p70 antagonist (34, 35) and inhibits DTH responses to soluble Ags (45). Hence, we used mice deficient in either the p35 or p40 subunit of IL-12 to further probe the requirement for biologically active IL-12p70 in alloimmunity and to evaluate the effects of endogenously produced p40 on alloantigen-reactive Th1 development. Since p35 is not secreted in the absence of p40 (36), cells obtained from p40-/- mice do not secrete either subunit of IL-12. In contrast, cells from p35-/- recipients are capable of secreting p40 at concentrations similar to that of IL-12 WT mice (40).

The key in vivo observations made in the current study are as follows. 1) Biologically active IL-12p70 was not required for in vivo Th1 development following cardiac transplantation, in that Th1 activity was readily demonstrable in p40-/- mice. Further, neutralizing endogenous IFN-{gamma} did not ablate Th1 responses in p40-/- allograft recipients, suggesting that alloreactive Th1 development may occur independent of both IL-12 and IFN-{gamma} in vivo. 2) While Th1 development in p35-/- allograft recipients was equivalent to that observed in IL-12 WT mice, Th1 responses in p40-/- mice were reduced. This observation suggests that in p35-/- mice, endogenously produced p40 may substitute for IL-12p70 in alloreactive Th1 development following transplantation. Indeed, neutralizing endogenous p40 in p35-/- allograft recipients reduced Th1 responses to the concentration observed in p40-/- mice (Fig. 1Go). This effect of endogenous p40 on Th1 stimulation was not apparent when splenocytes obtained from p35-/- and p40-/- mice were stimulated with either Con A (Table IGo) or alloantigens (Table IIGo) in vitro. This may be related to the concentration of p40 that is produced in tissue culture compared with the concentration achieved within a rejecting allograft, where 50% or more of the graft infiltrating cells may be inflammatory macrophages (46, 47). While the concentration of p40 within a rejecting allograft is not known, one might predict that the concentration is high at a site of inflammation that is rich in inflammatory macrophages. In further support of this possibility, Mauro et al. (48) reported that little or no IL-12 is produced during primary in vitro culture of lymphocytes with alloantigens. Our original observation that p40 homodimer stimulated alloreactive Th1 in vitro was made when MLC were supplemented with a high concentration (500 ng/ml) of p40 homodimer (38).

The in vitro Th1 response to either Con A (Table IGo) or alloantigens (Table IIGo) was markedly enhanced by IL-12p70, in that IL-12 WT splenocytes produced significantly more IFN-{gamma} than did p35-/- or p40-/- cells. Cells from IL-12-deficient mice were responsive to exogenous rIL-12, indicating that these cells expressed a functional IL-12R following antigenic stimulation. Further, the addition of anti-IL-12 Abs to Con A stimulated IL-12 WT splenocytes decreased Th1 function to the concentration seen in cultures of IL-12-deficient splenocytes (Table IGo). Collectively, these in vitro data are in keeping with previous reports that a major function of IL-12 is to augment Th1 development (Reviewed in Refs. 1 and 2).

However, Th1 responses were reproducibly present in the absence of IL-12 both in vitro (Tables I and II) and in vivo (Fig. 1Go), illustrating that IL-12 is not needed for the development of IFN-{gamma}-producing cells. Recent studies have shown that IFN-{gamma}, which is up-regulated by IL-12, is the critical cytokine in promoting and maintaining mouse Th1 function (31, 49). Interestingly, IFN-{alpha} is more active than IFN-{gamma} in maintaining human Th1 function (32). This Th1-promoting activity of IFN is related to the ability of this cytokine to induce IL-12Rß2 expression, and override the inhibitory effects of IL-4 on IL-12Rß2 expression (31, 32). Hence, we treated p40-/- cardiac allograft recipients with neutralizing anti-IFN-{gamma} mAb to determine whether Th1 development in the absence of IL-12 resulted from endogenous IFN-{gamma} produced independent of IL-12. However, alloantigen-reactive Th1 development was not dampened by neutralizing IFN-{gamma} in IL-12-deficient mice, suggesting that an alternate Th1-sensitizing pathway may be operative in vivo. A likely candidate is IL-18 or IFN-{gamma}-inducing factor, which has been reported to induce Th1 function independent of IL-12 (50). In addition to its ability to induce IFN-{gamma} production, IL-18 (but not IL-12) enhances anti-CD3-driven production of IL-2 and granulocyte-macrophage-CSF in vitro (51). To date, a role for IL-18 in alloimmune responses has not been established. However, we have found intragraft expression of IL-18 mRNA in recipients of IL-12 WT and IL-12-deficient allografts, as well as in syngeneic transplant recipients (J.R.P. and D.K.B., unpublished observation).

While Th1 development occurred in the absence of both IL-12 and IFN-{gamma}, the pathology of rejection differed from that observed when grafts are rejected in the absence of IL-12 alone (Fig. 3Go). Intravascular thrombosis formation was not observed when IL-12 WT cardiac allograft recipients were treated with anti-IFN-{gamma} mAb (data not shown), suggesting that the underlying mechanism responsible for this pathology may be suppressed by IL-12 and/or IFN-{gamma}. Despite the in vivo development of Th1 in p40-/- allograft recipients treated with anti-IFN-{gamma}, Th1 function (i.e., IFN-{gamma} production) was theoretically neutralized under these conditions. These conditions would favor the emergence of Th2-regulated effector mechanisms that may contribute to this alternate form of rejection (52). This possibility is currently under investigation.

Our in vitro (38) and in vivo (Fig. 1Go) observations that IL-12 p40 stimulates alloantigen-reactive Th1 may appear to contradict several reports that ascribe an inhibitory role for p40 in other models (34, 35, 45, 53, 54). For example, p40 inhibits IL-12p70 from binding to the IL-12R and inhibits IL-12-driven proliferation of mitogen-stimulated lymphoblasts (34, 45) and T cell lines (35). In these studies (34, 35), p40 homodimer was more effective than monomer at inhibiting IL-12-driven proliferation. In addition, treatment of mice with p40 homodimer inhibited both Th1 development and DTH responses to soluble Ags in vivo (45). However, responses to soluble Ags are predominantly mediated by CD4+ T cells (55). Indeed, we too have found that p40 homodimer inhibits CD4+ alloantigen-reactive Th1 responses both in vitro and in vivo (38). Hence, our observation that p40 stimulates CD8+ Th1 (38) (Fig. 2Go) is not in conflict with the study by Gately et al. (45). Rather, these studies may relate to a differential requirement for IL-12p70 in CD4+ vs CD8+ Th1 responses, as well as the potential for p40 to substitute for IL-12p70 in CD8+ Th1 development. However, Kato et al. (54) reported that the Th1 response to p40-transfected allogeneic myoblasts was reduced and that p40-expressing myoblasts survived several weeks longer than control cells in vivo. However, the effects of local p40 production on CD4+ vs CD8+ Th1 responses to allogeneic myoblasts were not reported (54). CD8+ cells dominate the Th1 response in p35-/- cardiac allograft recipients, in that depletion of CD8+ cells virtually eliminated the ability of splenocytes to produce IFN-{gamma} when restimulated with alloantigens in vitro (Fig. 2Go). Similarly, depletion of CD8+ cells markedly reduces Th1 function in IL-12 WT (38) and p40-/- (data not shown) cardiac allograft recipients. Differences in the effects of p40 reported by Kato et al. (54) and the current study may also be related to differences in the immune response to a nonvascularized cellular transplant, and the response to a vascularized cardiac allograft, which represents a complex tissue.

It is not clear why p40 functions as an IL-12p70 antagonist in some systems but appears to substitute for IL-12p70 in alloantigen-reactive CD8+ Th1 development. Since p40 binds IL-12Rß1 (26, 27), stimulatory effects of p40 on CD8+ Th1 would likely be mediated through this receptor component. Zou et al. (30) reported that IL-12Rß1 and IL-12Rß2 associate with different Janus kinases and therefore may contribute to distinct signaling pathways: the cytoplasmic domain of IL-12Rß1 associates with TYK2; while the cytoplasmic domain of IL-12Rß2 interacts with JAK2. Hence, IL-12Rß1 may be capable of transducing p40 signals via TYK2. To our knowledge, this possibility has not been tested. An alternate possibility is that IL-12Rß1 associates with an additional, as yet undefined component of the IL-12R on CD8+ but not CD4+ Th1. Regardless of an apparent functional distinction on CD4+ and CD8+, IL-12Rß1 is critical for IL-12-mediated signaling in that T cells and NK cells obtained from IL-12Rß1-deficient mice are not responsive to IL-12p70 (33).

In summary, our data illustrate that alloreactive CD8+ Th1 may develop in the absence of both IL-12 and IFN-{gamma} and that endogenously produced IL-12p40 may substitute for IL-12p70 in inducing optimal Th1 function. Further, the current study and other previously published reports (38, 45) suggest that IL-12p40 may serve as a useful IL-12R antagonist in the treatment of diseases that are regulated by CD4+ Th1 but not those disease states associated with CD8+ Th1.


   Acknowledgments
 
We thank Dr. Maurice K. Gately for generously providing anti-mouse IL-12 Abs and murine rIL-12 and for his helpful discussions.


   Footnotes
 
1 Financial support for this work was provided by Grant R01 AI31946 (D.K.B.) from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Joseph R. Piccotti, Transplant Immunology Research Laboratory, Section of General Surgery, A560 MSRB II, Box 0654, University of Michigan Medical Center, Ann Arbor, MI 48109-0654. Back

3 Abbreviations used in this paper: DTH, delayed-type hypersensitivity; IL-12p70, a 70- to 75-kDa heterodimer consisting of disulfide-bonded 35-kDa (p35) and 40-kDa (p40) subunits; p35-/-, IL-12 p35 knockout mice; p40-/-, IL-12 p40 knockout mice; IL-12 WT, IL-12 wild-type mice; H & E, hematoxylin and eosin. Back

Received for publication August 20, 1997. Accepted for publication October 16, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Dendritic Cell (DC)-Based Protection Against an Intracellular Pathogen Is Dependent Upon DC-Derived IL-12 and Can Be Induced by Molecularly Defined Antigens
J. Immunol., March 15, 2003; 170(6): 3171 - 3179.
[Abstract]