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The Journal of Immunology, 1999, 163: 1786-1792.
Copyright © 1999 by The American Association of Immunologists

Bruton’s Tyrosine Kinase Deficiency in Macrophages Inhibits Nitric Oxide Generation Leading to Enhancement of IL-12 Induction1

Sangita Mukhopadhyay*, Anna George*, Vineeta Bal*, Balachandran Ravindran{dagger} and Satyajit Rath2,*

* National Institute of Immunology, New Delhi, India; and {dagger} Regional Medical Research Center, Bhubaneswar, India


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
We show that macrophages of X-linked immunodeficient mice with a mutant nonfunctional Bruton’s tyrosine kinase produce less NO than wild-type macrophages in response to a variety of stimuli. Induction of the inducible NO synthase (iNOS) protein, the transcription factor IFN regulatory factor-1 involved in iNOS expression, and the transcription factor STAT-1 involved in regulating IFN regulatory factor-1 induction are all poorer in X-linked immunodeficient than in wild-type macrophages. On the other hand, induction of IL-12 is higher in X-linked immunodeficient than in wild-type macrophages. Macrophage IL-12 induction is enhanced by iNOS inhibitors such as aminoguanidine and thiocitrulline and is inhibited by NO generation via sodium nitroprusside. There is relative enhancement of IFN-{gamma} production by immune T cells from mice immunized under aminoguanidine cover. Our data thus suggest that Bruton’s tyrosine kinase participates in signaling for iNOS induction via IFN regulatory factor-1 in macrophages and that NO is an inhibitor of IL-12 induction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The cytokine effector molecules produced by effector T cells generated by immunization in vivo are centrally involved in optimal clearance of infections. Macrophage activating proinflammatory "type 1" cytokines such as IFN-{gamma} are essential for clearance of intracellular bacterial infections (1, 2), while "type 2" cytokines involved in B cell help such as IL-4 and IL-5 are more relevant for extracellular infections (3, 4). The differential commitment of responding T cells to producing either the type 1 (IFN-{gamma}, TNF-ß) or the type 2 (IL-4, IL-5, IL-10) cytokine groups (5, 6, 7) is regulated by both cognate and noncognate signals to T cells (8, 9, 10). Cytokine-mediated signals of APC origin are prominent among these. IL-6, IL-10, and IL-12 can all be produced by macrophages (11, 12, 13). While IL-6 and IL-10 drive T cells to the type 2 effector pathway (14, 15), IL-12 is the major APC cytokine known to drive them to IFN-{gamma} production as well as enhance T cell proliferation (16, 17, 18). All these APC-derived cytokines are inducible by a variety of environmental, potentially pathogen-derived stimuli (19, 20, 21). Thus, the induction of costimulatory cytokines on APCs appears to be a major factor in T cell priming, and the regulation of induction of these cytokines becomes an issue of interest in understanding how T cell responses are controlled.

This induction of costimulatory cytokines from APCs is commonly seen to occur in response to the same stimuli that lead to APC activation for effector purposes, such as macrophage activation for phagocytosis and pathogen clearance. Molecules induced during such effector activation of macrophages prominently include generators of free radicals such as NO (22, 23). The connections between the induction of costimulatory and effector molecules in activated APCs are likely to be a point of immunoregulatory control. Signal transduction events in APCs are one target of studies involving the regulation of induction of costimulatory and effector cytokines and their effects on each other (24, 25). Nonreceptor-associated tyrosine kinases have been shown to be important components of many signaling cascades. One such enzyme, Bruton’s tyrosine kinase (Btk),3 is expressed in both B cell and myeloid APC lineages (26, 27), but its functional significance has so far been examined mainly in the context of B cell activation (28, 29). We report here its role in regulating NO induction and IL-12 production in macrophages using mice carrying a mutation in the btk gene and demonstrate the cross-regulatory control of IL-12 production by NO.


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

Btk-mutant X-linked immunodeficient (xid) CBA/N mice and their wild-type counterpart CBA/J mice, obtained from The Jackson Laboratories (Bar Harbor, ME) and bred in the small animal facility of the National Institute of Immunology (New Delhi, India), were used for all experiments at 6–10 wk of age. All animal experimentation was done with the approval of the Institutional Animal Ethics Committee.

Immunization

Chicken conalbumin (CA; Sigma, St. Louis, MO) was used as the immunogen without adjuvant. Mice were immunized with 1 mg Ag in 0.5 ml PBS i.p. and sacrificed on days 7–10 postimmunization. Mice received the selective inducible NO synthase (iNOS) inhibitor aminoguanidine (AG; 1 mg in 0.2 ml PBS/mouse) i.p. 12 h before immunization where appropriate and every 12 h thereafter until the end of the experiment. Control groups received PBS alone.

T cell activation assays

Mice were sacrificed by cervical dislocation, and splenic cells were isolated for T cell response assays. Cells (3 x 105/well) were cultured in Click’s medium (Irvine Scientific, Santa Ana, CA) containing 0.05 mM 2-ME, 10% FCS (Life Technologies, Gaithersburg, MD) and antibiotics, along with titrated doses of CA in 200 µl per well at 37°C. Cytokine assays were done in culture supernatants after 60 h of incubation. Proliferation was assayed after 96 h of incubation by pulsing the cultures with 0.5 µCi [3H]thymidine per well for 10–12 h and then harvesting and counting the plates by scintillation spectroscopy (Betaplate, Pharmacia-Wallac, Turcu, Finland). All assays were done in triplicate cultures, and data is expressed as mean cpm ± SE.

Macrophage stimulation assays

Peritoneal exudate cells (PECs) were induced by injection of thioglycollate broth. Peritoneal macrophages were isolated by plastic adherence and harvested by vigorous flushing with medium. Adherent PECs were cultured in 96-well plates at 2–4 x 105 per well in the presence of titrated doses of either bacterial LPS (Salmonella typhosa; Sigma), IFN-{gamma} (Genzyme, Boston, MA) or anti-CD-40 (PharMingen, San Diego, CA). The NOS inhibitors AG or thiocitrulline (TC) were added 30 min before stimulator addition where used. Nitrite accumulation and IL-12 secretion in the culture supernatants were measured after 48 h of incubation.

Cytokine assays

IL-12, IL-10, and IFN-{gamma} were measured in culture supernatants by two-site sandwich enzyme-linked immunoassays (EIA; Duoset, Genzyme, and PharMingen). Standard curves for the cytokines were obtained using the recombinant standard proteins provided by the manufacturers.

Nitrite estimation

The accumulated nitrite resulting from NO production by the stimulated macrophages in culture was measured using the Griess reaction (30). Briefly, 100 µl of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine (1:1) in orthophosphoric acid) was added to 100 µl of culture supernatants. Absorbance was measured at 550 nm. The nitrite content in the samples was calculated based on a standard curve read from a prepared standard solution of sodium nitrite.

Western blot analyses for iNOS, IFN regulatory factor-1 (IRF-1), and STAT-1

Western blot analysis was used to detect cellular iNOS protein levels as well as nuclear IRF-1 and STAT-1 levels. For iNOS detection, LPS-stimulated (10 µg/ml, 48 h) or nonstimulated CBA/N or CBA/J macrophages were lysed as described (31), dialysed, and stored at -70°C. Nuclear extracts from macrophage cultures were prepared from Nonidet P-40-lysed cells as described (32). Protein concentrations were estimated using the bicinchoninic acid method (Pierce, Rockford, IL). Cell lysates or nuclear extracts containing 50 µg protein were separated by SDS-PAGE in a 10% running gel and electroblotted (Bio-Rad, Hemel Hampstead, U.K.) onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). The membranes were stained with 0.5 µg/ml of affinity-purified rabbit Abs to iNOS, IRF-1, or STAT-1 (Santa Cruz Biotechnology, Santa Cruz, CA) followed by donkey anti-rabbit IgG-HRP (Sigma) using published protocols (33). Bound enzyme was detected by chemiluminescence following the manufacturer’s protocols (Amersham, Little Chalfont, U.K.). The profiles were scanned and analyzed densitometrically using NIH Image software.

Statistical analysis

Data were analyzed using Student’s t test where appropriate.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
The Btk molecule has been shown to be involved in a wide range of signal transduction processes in B cells (34, 35, 36, 37). The expression of Btk in myeloid cells and monocytes is well known (26, 27), although there are fewer data documenting its role in signaling in non-B cell populations. Mast cell signaling via Fc{epsilon} receptors has been shown to involve Btk (38), raising the possibility that other myeloid cell types may also use it. We have found that CBA/N mice tend to generate relatively more type 1 cytokine responses upon immunization, and that this tendency is macrophage controlled. Type 1 and type 2 T cell clones have been reported to be differentially sensitive to NO-mediated inhibition of activation (39). Mice engineered to be iNOS-deficient show anti-Leishmania T cell responses higher in proliferative magnitude and with a relative dominance of type 1 cytokines as well as of the IFN-{gamma}-dependent IgG2a isotype in serum anti-Leishmania Abs (40). There are suggestions that the induction of iNOS (41) may be dependent on tyrosine phosphorylation (42). Therefore, we began by examining the efficiency of NO generation by various stimuli in CBA/N macrophages deficient in Btk.

Macrophages in CBA/N mice are deficient in NO induction

The assay used was an estimation of the nitrite accumulated upon oxidation of NO in the culture medium. Induced generation of NO leading to accumulation of nitrite is dependent on the induction of iNOS in macrophages (41, 43). The stimuli we have used are bacterial LPS as a pathogen-derived stimulator, IFN-{gamma} as an endogenous activator of macrophages during an immune response and anti-CD40. There is evidence that at least some signals mediated through CD40 on B cells are independent of the CBA/N mutation (44), although there are other reports that all signaling consequences of CD40 ligation may not be intact in them (45, 46). Because CD40 is one of the signal transducing cell surface molecules shared by B cells and macrophages, we have used anti-CD40 as a stimulus for examining CBA/N macrophage defects.

Fig. 1Go shows that CBA/N macrophages accumulated significantly reduced amounts of nitrite than CBA/J macrophages did in response to all stimuli used by a factor of 4- to 10-fold. In multiple independent experiments, the dose of LPS needed to generate equivalent induction of nitrite accumulation went up from 1.0 ± 0.06 µg/ml for CBA/J cells to 9.6 ± 0.9 for CBA/N cells (mean ± SD; p < 0.001). The dose of IFN-{gamma} similarly needed went up from 5.2 ± 4.3 ng/ml to 27.7 ± 2.1 ng/ml, while the dose of anti-CD40 mAb required went up from 0.21 ± 0.13 µg/ml to 0.93 ± 0.05 µg/ml (mean ± SD; p < 0.01).



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  		FIGURE 1. Induction of nitrite accumulation in macrophage culture is poorer in CBA/N compared with CBA/J mice. Peritoneal macrophages from CBA/J (open symbols) or CBA/N (filled symbols) mice were cultured with varying doses of either bacterial LPS (A), IFN-{gamma} (B), or anti-CD40 (C) in triplicate cultures with (squares) or without (circles) the iNOS inhibitor, AG (100 µg/ml). After 48 h, nitrite accumulation was measured in the culture supernatants and expressed as µM (mean ± SE). Levels of nitrite in nonstimulated macrophage supernatants were in the range of 0–4 µM. The results are representative of six independent experiments.

 
In addition to suggesting that Btk may be involved in NO generation pathways, this also shows that at least some CD40-mediated signals are indeed deficient in btk-mutant macrophages as well. We have used a selective inhibitor of iNOS, AG (47), to establish that the nitrite accumulation in these cultures is a consequence of iNOS induction. The addition of AG to these cultures inhibited the nitrite levels completely no matter which stimulus was used (Fig. 1Go). These data suggest that iNOS induction is compromised in CBA/N macrophages, and therefore Btk is likely to play a significant role in the signal transduction cascade responsible for iNOS induction. The reduction in the ability of stimuli as varied as LPS and IFN-{gamma} to induce nitrite accumulation from CBA/N macrophages also suggests that Btk is likely to be involved in a region of the iNOS induction pathway common to many disparate stimuli. However, the fact that AG further decreased the nitrite accumulation induced even from CBA/N macrophages also suggests that at least some degree of iNOS induction is possible via pathways that are either Btk independent or at least do not require the plekstrin homology domain of Btk, which is mutated in CBA/N mice (48).

Reduction in the induction of iNOS, IRF-1, and STAT-1 in CBA/N macrophages

We next examined the induction of iNOS in these macrophage cultures directly using polyclonal anti-iNOS Abs in Western blot assays. Cytosolic extracts of macrophages from either CBA/J or CBA/N mice activated with 10 µg/ml LPS in culture were probed 48 h later in Western blots for iNOS protein. Fig. 2Go shows that, while no significant levels of iNOS protein were detectable in resting macrophages from either strain, CBA/J macrophages showed a stronger induction of iNOS than CBA/N macrophages did upon activation (difference of 2- to 3-fold by densitometry in multiple experiments). These data are consistent with the earlier data showing poorer generation of NO in activated CBA/N macrophages and directly implicate Btk in the regulation of signaling for iNOS induction.



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  		FIGURE 2. Induction of expression of iNOS, IRF-1, and STAT-1 is poorer in CBA/N macrophages compared with CBA/J macrophages. Peritoneal macrophages from either CBA/J or CBA/N mice were stimulated with 10 µg/ml of bacterial LPS or left nonstimulated, and 48 h later the expression of either iNOS in cell lysates or IRF-1 and STAT-1 in nuclear extracts was examined by Western blot analysis (50 µg total protein/well). The data shown are representative of three separate experiments.

 
A number of transcriptional activators have been analyzed for their roles in transcriptional regulation of iNOS (49, 50, 51, 52, 53). While activators such as members of the rel family of proteins (51, 53) have been implicated in iNOS regulation, the clearest evidence so far suggests that IRF-1 is a major crucial factor in iNOS induction (49, 50). Therefore, we examined the induction of nuclear IRF-1 in CBA/N vs CBA/J macrophages activated by LPS. Fig. 2Go shows Western blot analysis of nuclear extracts from nonstimulated or stimulated macrophages of either strain probed with a polyclonal anti-IRF-1 Ab. While resting macrophages show very little nuclear IRF-1, activated macrophages show excellent induction, and, again, CBA/N macrophages are notably poorer in IRF-1 induction than CBA/J macrophages (difference of 3- to 5-fold by densitometry in multiple experiments). Therefore, it is possible that the contribution of Btk in NO generation is mediated through IRF-1 induction. Further, the transcription factor STAT-1 has been reported to be an inducer of IRF-1 transcription (54). Therefore, the LPS-mediated induction of STAT-1 in the nucleus was also assayed in unstimulated and LPS-activated CBA/J or CBA/N macrophages. As shown in Fig. 2Go, STAT-1 induction is also poor in CBA/N macrophages as compared with CBA/J macrophages (difference of 2- to 4-fold by densitometry in multiple experiments).

It is noteworthy that CBA/N cells show only a partial reduction of iNOS, IRF-1, and STAT-1 induction rather than a complete block. This raises a number of possibilities, such as Btk being part of either a nonessential biochemical pathway that can nonetheless contribute to enhance STAT-1/IRF-1/iNOS induction synergistically, or other tyrosine kinases being able to substitute to some extent for Btk in the main STAT-1/IRF-1/iNOS induction pathway arguing for some degree of redundancy of Btk function, or the mutation in the plekstrin homology domain of Btk seen in the xid strain only partially abrogating this particular function of Btk.

IL-12 expression in CBA/N macrophages and the role of NO in IL-12 regulation

There have been demonstrations of enhanced anti-Leishmania Th1 immunity in iNOS-deficient mice (40) and suggestions that iNOS may contribute to the generation of a Th2 response via NO (39). Therefore, we next looked at the ability of CBA/N macrophages to generate IL-12. CBA/N and CBA/J macrophages in culture were stimulated with bacterial LPS, IFN-{gamma}, or anti-CD40 as shown in Fig. 3Go, with or without the addition of the selective iNOS inhibitor AG (100 µg/ml), and the IL-12 levels produced by these cells were estimated in the culture supernatants. The results show (Fig. 3Go) that CBA/N macrophages produced greater levels of IL-12 in culture than CBA/J cells, suggesting a role for Btk in regulating IL-12 production. In multiple independent experiments, the reduction in the ability of CBA/N macrophages to produce IL-12 was increased by a factor of 4- to 10-fold. The dose of LPS needed to generate equivalent induction of IL-12 went down from 7.8 ± 1.4 µg/ml for CBA/J cells to 1.1 ± 0.2 for CBA/N cells, the dose of IFN-{gamma} similarly needed decreased from 26.8 ± 0.9 ng/ml to 1.0 ± 0.1 ng/ml, while the dose of anti-CD40 mAb required went down from 0.8 ± 0.04 µg/ml to 0.1 ± 0.01 µg/ml (p < 0.001).



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  		FIGURE 3. IL-12 induction is poorer in CBA/J than in CBA/N macrophages and iNOS inhibition enhances the expression of IL-12. Peritoneal macrophages from CBA/J (circles) or CBA/N (squares) mice were cultured with varying doses of the stimuli shown with (filled symbols) or without (open symbols) 100 µg/ml of AG in triplicate cultures. After 48 h of stimulation, both IL-12 production and nitrite accumulation were measured in the culture supernatants. Results shown are representative of five independent experiments.

 
Further, inhibition of iNOS and nitrite generation by AG in these cultures enhanced the levels of IL-12 produced (Fig. 3Go). Thus, in multiple independent experiments, AG reduced the dose of LPS required for equivalent IL-12 production by 7- to 10-fold for CBA/J cells and by 4- to 6-fold for CBA/N cells (p < 0.001). Similarly, the dose of IFN-{gamma} needed was reduced by 15- to 25-fold for CBA/J cells (p < 0.001) and by 3- to 5-fold for CBA/N cells (p < 0.05), and the reduction in the dose of anti-CD40 mAb was 5- to 7-fold for CBA/J macrophages (p < 0.001) and 4- to 6-fold for CBA/N macrophages (p < 0.01). These data support a causal link between iNOS/NO and IL-12 expression and suggest one possible way by which Btk influences IL-12 induction.

If the iNOS/NO pathway is one major route by which Btk influences IL-12 induction, IL-12 production in CBA/J cells would be expected to be more sensitive to NOS inhibition than in CBA/N cells. Therefore, we next examined the sensitivity of CBA/N and CBA/J macrophages to NOS inhibitor-mediated up-regulation of IL-12 using bacterial LPS (10 µg/ml) as the stimulus and titrated doses of either AG or TC, another competitive inhibitor of NOS that affects all forms of NOS, unlike AG, which has been known to inhibit iNOS in relatively select fashion (47, 55). Fig. 4GoA shows the enhancement of IL-12 induction caused by AG or TC in CBA/J and CBA/N macrophages. TC behaved exactly the same way that AG did in causing a dose-dependent enhancement of IL-12 induction. When the degree of enhancement is plotted as normalized fold-enhancement (Fig. 4GoB), it is evident that IL-12 induction in CBA/N macrophages does in fact respond far more poorly to NOS inhibitors than CBA/J macrophages. Thus, in multiple independent experiments, the dose of AG needed for a 2-fold enhancement of IL-12 induction was 97 ± 64 µg/ml for CBA/J macrophages, but it went up to 651 ± 142 µg/ml for CBA/N cells (mean ± SD; p < 0.01).



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  		FIGURE 4. CBA/J macrophages are more sensitive than CBA/N macrophages to enhancement of LPS-mediated induction of IL-12 expression by NOS inhibitors. A, The levels of IL-12 as measured by EIA in the culture supernatant of peritoneal macrophages from either CBA/J (open symbols) or CBA/N (filled symbols) mice, stimulated with bacterial LPS for 48 h in presence of titrated doses of either AG (circles) or TC (squares), are shown. Levels of IL-12 in LPS-activated macrophage culture supernatants in the absence of any NOS inhibitor were 1311 pg/ml for CBA/N cells and 845 pg/ml for CBA/J cells. The data in A are recalculated in B to show the fold enhancement in IL-12 induction by AG or TC in CBA/J or CBA/N macrophages. Results shown are representative of three independent experiments.

 
Together, these data suggest that one major route by which Btk influences IL-12 production is likely to be through the NO-mediated pathway. This is also of significance to the physiological role of many of the transcriptional activators implicated in driving either iNOS and/or IL-12, because many of the transcriptional activators thought to play a role in the regulation of iNOS, such as the rel family of proteins (although not IRF-1), are also thought to be involved in IL-12 transcription (56, 57).

Exogenous NO inhibits IL-12 induction in macrophages

The data so far suggest that endogenous NO generation via iNOS has an inhibitory influence on IL-12 induction. To confirm this independently, we next attempted to assay the influence of exogenously administered NO on IL-12 induction in macrophages. Bacterial LPS was used (10 µg/ml) as the inducing stimulus for CBA/N and CBA/J macrophages, and AG (300 µg/ml) was used to inhibit all endogenous iNOS and therefore prevent any endogenous NO production from being triggered by LPS. Titrated doses of sodium nitroprusside (SNP) were added to the cultures as an exogenous generator of NO. Fig. 5GoA shows that the levels of induced IL-12 fell steadily with increasing doses of SNP. Clearly, no matter what the source, NO in macrophages is inhibitory for IL-12 production. Under these circumstances, where endogenous NO production was eliminated, CBA/J and CBA/N macrophages behaved similarly and were equally sensitive to SNP-mediated inhibition of IL-12 induction (Fig. 5GoB), so that while the concentration of SNP needed for 50% inhibition of the IL-12 induction was 400 ± 82 µM for CBA/J macrophages, it was 478 ± 88 µM for CBA/N macrophages (p > 0.1) in multiple independent experiments. These data suggest that the effect of Btk in regulating IL-12 induction through the iNOS/NO pathway is likely to be mediated mainly via regulation of iNOS induction rather than through any differential sensitivity of IL-12 induction to NO itself.



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  		FIGURE 5. The NO generator SNP inhibits LPS-mediated induction of IL-12 in both CBA/J and CBA/N macrophages. Peritoneal macrophages from either CBA/J (open circles) or CBA/N (filled circles) mice were stimulated with 10 µg/ml of bacterial LPS in the presence of 100 µg/ml AG to inhibit endogenous NO generation and titrated concentrations of SNP. After 48 h, IL-12 was measured in the culture supernatants by EIA (A). Levels of IL-12 in AG-inhibited and LPS-activated macrophage culture supernatants in the absence of any SNP were 895 pg/ml for CBA/N cells and 793 pg/ml for CBA/J cells. The data in A are recalculated in B to show the percentage inhibition of IL-12 induction by SNP in CBA/J or CBA/N macrophages. Results shown are representative of two independent experiments.

 
Curiously, although macrophages from peritoneal resident cells of both CBA/J and CBA/N mice produce very little nitrite upon stimulation compared with PEC macrophages, they produce IL-12 at levels comparable to those from PEC macrophages of the respective strains (data not shown), suggesting that the iNOS-mediated pathway is likely to be an extraneous modulator of IL-12 production rather than being crucial for it. However, the mechanism of action of NO in the regulation of IL-12—transcriptional, posttranscriptional, or transportational—is not addressed by our data so far.

AG mimics the Btk-mutant effect on T cell responses in vivo

Induced macrophage NO has pleiotropic effects (58). Many of these effects have been argued to be in its capacity as an "effector" molecule (22, 23), and data in a variety of experimental systems using iNOS-deficient mice have been interpreted thus. Therefore, given our data, it was of obvious interest to ask if the effect of NO on IL-12 production could actually play a regulatory role in controlling both the magnitude and the cytokine balance of T cell responses in normal mice in vivo.

Therefore, wild-type CBA/J mice were immunized with CA under cover of AG, and T cell responses were assayed at 8 days postimmunization. Fig. 6GoA shows that there is an increase in the magnitude of the T cell proliferative response if immunization has been done under AG cover. The cytokine balance (IL-10/IFN-{gamma} ratio) in AG-treated mice shifts significantly in favor of Th1 responses (Fig. 6GoC; p < 0.01), with decreases in IL-10 levels and increases in IFN-{gamma} levels (Fig. 6GoB). Thus, iNOS induction (and NO production) in vivo has a significantly dominant regulatory role for selection of T cell response pathways, as predicted by our data and by previous studies with iNOS-deficient mice (40).



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  		FIGURE 6. AG, an iNOS inhibitor, enhances both the relative type 1 cytokine component and the magnitude of the T cell response in vivo. Responses of spleen cells from CBA/J mice with (filled symbols) or without (open symbols) AG treatment during immunization with CA were measured at day 8 postimmunization. A, The magnitude of proliferative responses of spleen cells to titrated doses of CA is shown. B, The levels of IL-10 (circles) and IFN-{gamma} (squares) observed are shown. C, The IL-10/IFN-{gamma} ratios (mean ± SE) obtained in the supernatants from these activated T cell cultures are shown. Results shown are representative of five independent experiments.

 
We have already shown that CBA/N mice show a far greater tendency to mount Th1-dominated T cell responses to immunization, and that this tendency can be modulated by adoptive transfer of CBA/J macrophages, but not B cells, before immunization (59). The present data now suggest that the ability of CBA/N macrophages to produce higher levels of IL-12 may contribute to this Th1-dominated characteristic of CBA/N mice. Further, we show that Btk-deficient macrophages show defective induction of NO production via iNOS, probably via lack of optimal induction of the transcription factor IRF-1 to which a block in the induction of STAT-1 may contribute, and that the NO metabolites inhibit IL-12 induction in macrophages in their turn significantly enough to affect the type 1/type 2 T cell cytokine balance in vivo.

It thus appears that a macrophage effector molecule, NO, inhibits the induction of a macrophage costimulatory cytokine, IL-12, pointing to the possibility of a feedback inhibitory loop down-modulating immune responses once effective effector responses are achieved. Interestingly, we find in preliminary experiments that, in common with iNOS, the induction of other macrophage effector cytokines such as TNF-{alpha} and IL-1ß is also poorer in CBA/N than in CBA/J macrophages (data not shown). This suggests that the effect of NO on induction of various macrophage products is not a nonspecific down-regulation. Further, in conjunction with our data showing that induction of high levels of expression of various costimulatory macrophage surface molecules is also equivalent between CBA/J and CBA/N cells (data not shown and Ref. 59), the present data suggest a possible dichotomy of regulation between "T cell priming" vs "effector" products of macrophages. Coupled to recent data showing that iNOS is required for the effects of IL-12 in innate immunity such as macrophage and NK cell activation (60), these data may also imply the existence of hierarchies of cross-talk so that while IL-12 induces and to some degree works through iNOS (60), iNOS in turn helps down-regulate IL-12.

However, it must be pointed out that it is not clear if iNOS is induced in macrophages in vivo during the early stages of an immune response. Therefore, it is still possible that early IL-12 production from macrophages in vivo is not modulated through iNOS, despite our finding that both IL-12 and the Th1/Th2 balance are modified by AG. Thus, it still remains possible that IL-12 inhibition may not be the only pathway by which iNOS/NO modulate the Th1/Th2 balance. Nonetheless, the data presented here suggest the existence of intricate regulatory systems deciding the bioeffective molecules that would be produced by an activated macrophage. Detailed characterization of the signal transduction pathways involved in these multiple interactions we demonstrate would be of great interest in the generation of pharmacological modulators of immune responses.


   Acknowledgments
 
We thank Drs. A. Rudensky, C. A. Janeway, J. M. Durdik, R. Sen, S. Pillai, and Y. Liu for discussions. We thank Drs. R. K. Anand and R. K. Juyal for advice on breeding and maintenance of mice, and Sudip Ghosh for help with nuclear lysate preparations and Western blot analyses.


   Footnotes
 
1 This project was partly supported by grants to V.B. and A.G. from the Department of Science and Technology, Government of India. The National Institute of Immunology is supported by the Department of Biotechnology, Government of India. The Regional Medical Research Center is supported by the Indian Council of Medical Research. Back

2 Address correspondence and reprint requests to: Dr. Satyajit Rath, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110 067, India. E-mail address: Back

3 Abbreviations used in this paper: Btk, Bruton’s tyrosine kinase; AG, aminoguanidine; CA, chicken conalbumin; EIA, enzyme-linked immunoassay; iNOS, inducible nitric oxide synthase; IRF-1, IFN regulatory factor-1; PEC, peritoneal exudate cell; SNP, sodium nitroprusside; TC, thiocitrulline; xid, X-linked immunodeficient. Back

Received for publication December 10, 1998. Accepted for publication May 28, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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