Introduction

Top Abstract Introduction Materials and methods Results Discussion References

Interleukin-12 (IL-12) is a heterodimeric 70-kD (p70) cytokine composed of two covalently linked, glycosylated, 40-kD (p40) and 35-kD (p35) chains (1). IL-12 is mainly produced by monocytes, macrophages, and dendritic cells in response to bacterial products such as lipopolysaccharides (LPS), to intracellular pathogens (2, 3) or upon interaction with activated T cells (4, 5). IL-12 was originally discovered because of its ability to induce interferon- (IFN-) production, cell proliferation, and cytotoxicity mediated by natural killer cells and T cells (2, 6). It is now well established that IL-12 also plays a key role in the development of Th1 responses, leading to IFN- and IL-2 production (3, 7). These cytokines can in turn promote T-cell responses and macrophage activation.

Although only the covalently linked p70 heterodimer of IL-12 (IL-12 p70) has biologic activity, the p40 heavy chain is usually secreted in large excess over the p70 heterodimer, whereas free p35 has not been detected in supernatants of culture cells (2). Several studies have postulated the inducibility of both p35 and p40 in human monocytes by LPS (9, 10). Other studies, however, have suggested that only p40 is highly regulated by LPS, whereas p35 is constitutively expressed at low levels in peripheral blood mononuclear cells (2, 11). Therefore, depending on the cell type and the kind of stimuli, p35 and p40 mRNA expression may be regulated differently.

IL-12 is a pivotal cytokine in determining the nature and efficacy of immune responses, and understanding the endogenous mechanisms that modulate its production is important. Several cytokines regulate the ability of human antigen-presenting cells to produce IL-12. IFN--priming of monocytes/macrophages increases their ability to produce IL-12 (9, 12, 13), whereas IL-10 strongly inhibits it (14, 15). Several recent reports have shown that IL-12 may induce IL-10 production by T cells (16), which suggests a potential mechanism of negative feedback regulation of the immune response. To clarify and extend these findings to human macrophages, we compared in this report the capacity of LPS-stimulated human pulmonary alveolar macrophages (AM) to produce IL-12 p40 and IL-12 p70 with that of peripheral blood monocytes. The role of IFN- and IL-10 to up- or downregulate LPS-induced IL-12 p40 and IL-12 p70 production in AM was investigated. With reverse transcription-polymerase chain reaction (PCR), we showed that p35 is constitutively expressed on AM, whereas both p35 and particularly p40 mRNA are upregulated by LPS, alone or in association with IFN-. Furthermore, as LPS-stimulated macrophages released not only IL-12 but also IL-10, we demonstrated the strong inhibitory role of autocrine IL-10 on IL-12 secretion by LPS-stimulated AM but not by CD40-stimulated AM.

	Materials and Methods

Top Abstract Introduction Materials and methods Results Discussion References

Reagents and Media

RPMI 1640 medium containing penicillin (100 U/ml), streptomycin (100 U/ml), glutamate (10 mg/ml), heat-inactivated (HI) fetal calf serum (FCS) (10%), and Hanks' balanced salt solution was obtained from Gibco (Paisley, Scotland), Ficoll-Paque was obtained from Pharmacia Biotech (Uppsala, Sweden). Human IFN- (6 × 10⁶ U/ml) was purchased from Boehringer Ingelheim (Basel, Switzerland). LPS (Escherichia coli 026:B6) was from Difco (Detroit, MI). Recombinant human (rh)IL-4 was purchased from R&D Systems (Minneapolis, MN); rhIL-1 and rhIL-1 were obtained from Glaxo IMB (Geneva, Switzerland); recombinant human tumor necrosis factor- (rhTNF-) was from Biogen SA (Geneva, Switzerland); and rhIL-10 was purchased from Immugenex (Los Angeles, CA). Anti-IL-10 monoclonal antibody (mAb) was kindly provided by DNAX (Palo Alto, CA) (20). Anti-CD40 mAb (mAb 89) was from Immunotech (Marseille, France). CD32-transfected L cells were kindly provided by Dr. J. Banchereau (Schering-Plough, Dardilly, France). Primers for IL-12 p35 and p40 were synthesized by Microsynth (Marseille, France).

Preparation of AM and Monocytes

AM were obtained as previously described by bronchoalveolar lavage from a surgical specimen at a distance from a pulmonary cell carcinoma (21). Monocytes (Mo) were isolated from buffy coat of normal donors after Ficoll- Hypaque gradient sedimentation and purified by adherence after 1 h of culture with RPMI 1640 with 10% HI FCS. Adherent cells, harvested with a rubber policeman, consisted of 95% Mo, as assessed by differential count on May-Grünwald-Giemsa-stained smears.

To establish kinetics, AM and Mo were cultured in RPMI 1640 medium with 10% HI FCS at a final concentration of 1 × 10⁶ cells/ml in 24-well tissue culture plates, 16 mm in diameter (Costar, Cambridge, MA) for different periods of time. Cells were either unstimulated, treated with LPS (10 µg/ml) or IFN- (1,000 U/ml), or stimulated with both LPS and IFN-. AM and Mo were also stimulated by LPS (10 µg/ml) and a dose-response of IFN- (1 to 1,000 U/ml), or by LPS and IFN- in the presence of rhIL-10 (50-5,000 pg/ml) for 24 h. Culture supernatants were harvested and used for detection of IL-12 p40, IL-12 p70, and IL-10. In some experiments, AM were stimulated by anti-CD40 mAb immobilized on CD32-transfected L cells to induce IL-10 and IL-12 production, as previously described by Rousset and colleagues (22).

IL-10 and IL-12 Determination

IL-10 was measured by enzyme-linked immunosorbent assay (ELISA) with a BioSource Cytoscreen kit (Camarillo, CA); the limit of detection of the kit was 5 pg/ml. IL-12 p40 and IL-12 p70 were measured by ELISA with specific R&D Quantikine kits (R&D Systems); the limit of detection of the kits was 15 pg/ml and 5 pg/ml, respectively.

Reverse Transcription-PCR Analysis of IL-12

Modulation of IL-12 p35 and IL-12 p40 mRNA expression was assayed by PCR. RNA was prepared using a commercial modification of Chomczynski's method (23) (Trizol) from AM stimulated or not at different times by IFN- (1,000 U/ml), LPS (10 µg/ml), or by addition of both reagents. One microgram of total RNA was reverse transcribed using 0.5 µg oligo(dT) 15 primers (Boehringer Mannheim, Mannheim, Germany), 1 mM of each dNTP, 50 U Expand reverse transcriptase of Superscript (Boehringer Mannheim) transcriptase, and 1 U rRNasin (Promega, Madison, WI), in 20 µl for 45 min at 42°C. One twentieth of the reverse transcription (RT) reaction mixture was then amplified in a thermocycler (Perkin-Elmer-Cetus 9600; Norwalk, CT). Amplification of IL-12 p40 cDNA (24) and p35 (25) was done in a volume of 20 µl, with 0.2 U of Taq polymerase, 100 µM of each dNTP, and 400 nM of each 5' and 3' primer for 40 cycles. The cycling conditions were 0.8 min at 94°C, 0.8 min at 62°C, and 0.8 min at 72°C. Amplification of -actin cDNA was done in a volume of 20 µl, with 0.2 U of Taq polymerase, 200 µM of each dNTP, and 400 nM of each 5' and 3' primer (CLONTECH Laboratories, Inc., Palo Alto, CA) for 35 cycles. The cycling conditions were 45 s at 94°C, 45 s at 60°C, and 2 min at 72°C. The primers were synthesized by Microsynth (Balgach, Switzerland) and have the following sequences: (1) the -actin-primers 5':ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG and 3':CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC, (2) the IL-12 p40 primer 5':CCA AGA ACT TGC AGC TGA AG and 3':TGG GTC TAT TCC GTT GTG TC, and (3) the IL-12 p35 primers 5' AAC TAA TGG GAG TTG CCT GG and 3' GGA CCT CGC TTT TTA GGA AG. The predicted sizes of the PCR products were -actin, 838 bp; IL-12 p40, 354 bp; and IL-12 p35, 358 bp. An aliquot of PCR product was then visualized after electrophoresis through 2% agarose gels by ethidium bromide staining.

Statistical Analysis

Statistical analysis was performed using the Statview 4.05 program (Abacus Concepts, Berkeley, CA). Unpaired Student's t test was used to compare the production of IL-12 by AM and Mo. The Wilcoxon signed rank test was used to compare the inhibitory effects of several cytokines on IL-12 p70 production. Student's t test was used for the other experiments. Values of P < 0.05 were considered significant.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

Induction of IL-12 p35 and IL-12 p40 Expression in AM by LPS, IFN-, or LPS and IFN- Stimulation

To compare the capacity of LPS, IFN-, or LPS and IFN- stimulation to induce the expression of IL-12 p35 and IL-12 p40 subunits in AM, the pattern of expression of p35 and p40 mRNA was examined at different times of stimulation by using RT-PCR amplification. As shown in Figure 1, no p40 mRNA expression was detectable in AM cultured in medium alone, whereas a weak and transitory signal was observed for p35 mRNA expression at 4 h and 8 h. LPS stimulation increased the p40 mRNA level, which was faintly detectable at 4 h, and its expression further increased at 24 h. p35 mRNA expression was clearly present at 4 h and decreased between 15 h and 24 h. IFN- stimulation did not induce p40 mRNA expression but upregulated the expression of p35 mRNA. LPS and IFN- stimulation induced a progressive increase of p40 mRNA, its expression being maximal at 24 h, whereas maximal expression of p35 mRNA was already present at 4 h and remained at the same level thereafter.

View larger version (29K): [in this window] [in a new window]   Figure 1.   Kinetics of mRNA expression for IL-12 p35 and IL-12 p40 in AM. Human AM (1 × 107 cells/10 ml) were stimulated by LPS (10 µg/ml), IFN- (1,000 U/ml), or both LPS and IFN- for different periods of time. The expression of mRNA for IL-12 p35, IL-12 p40, and -actin was determined with RT-PCR. The resulting cDNA fragments were visualized after electrophoresis on 2% agarose gel. All the fragments had the expected lengths, as described in MATERIALS AND METHODS. The production of IL-12 p70 induced by the different stimuli was measured in the culture supernatants by ELISA. ND = not detectable (< 5 pg/ml). Results are representative of two distinct experiments.

Kinetics of IL-12 p40 and IL-12 p70 Production in AM and Mo

To determine the optimal conditions for IL-12 p40 and IL-12 p70 production by AM and Mo, cells were stimulated by LPS (10 µg/ml), IFN- (1,000 U/ml), or by both LPS and IFN- for different periods of time. No detectable amounts of cytokines could be measured in the culture supernatants of Mo or AM at 0 h (data not shown). As shown in Figure 2A, a significant amount of IL-12 p40 secretion was measured in the supernatants of Mo cultured in medium alone at all time points, but not in the supernatants of AM cultured in the same conditions. LPS increased the level of IL-12 p40 twofold in Mo, and induced IL-12 p40 production in AM 16 h after stimulation (Figure 2B). LPS-induced IL-12 p40 production in Mo reached a plateau 16 h after stimulation, whereas IL-12 p40 production in AM was slightly increased between 16 h and 48 h after stimulation. The level of LPS-induced IL-12 p40 in AM, however, was 6- to 8-fold lower than that induced in Mo. In contrast to IL-12 p40, IL-12 p70 was not induced in Mo and AM by LPS alone. IL-12 p40 production in IFN-- stimulated Mo was similar to medium alone, and IL-12 p70 production was not induced. IFN- stimulation did not induce IL-12 p40 or IL-12 p70 production in AM (data not shown). The addition of IFN- during LPS stimulation further increased IL-12 p40 production and induced IL-12 p70 production in both AM and Mo (Figure 2C).

View larger version (34K): [in this window] [in a new window]   Figure 2.   Kinetics of IL-12 p40 and IL-12 p70 production by human AM and Mo. Human AM and Mo (1 × 106 cells/ml) were cultured in medium alone (A), stimulated by LPS alone (10 µg/ ml) (B), or stimulated by LPS and IFN- (1,000 U/ml) (C) for different periods of time. The production of IL-12 p40 and IL-12 p70 was measured in the culture supernatants by ELISA. Results are expressed as means ± SEM (n = 6).

LPS and IFN- stimulation increased the level of IL-12 p40 in AM by 4- to 5-fold over stimulation by LPS alone, and only 1- to 2-fold in Mo. Both AM and Mo stimulated by LPS and IFN- released IL-12 p70 16 h after stimulation. The level of IL-12 p70 was still slightly increased between 16 h and 48 h after stimulation in AM, but not in Mo. In the following experiments, IL-12 p40 and IL-12 p70 production were analyzed after 24 h of stimulation, as Mo had reached their maximal secretion and as AM showed only a slight increase for both p40 and p70 protein production at 48 h compared with 24 h.

IFN- Increased LPS-Induced IL-12 p40 and IL-12 p70 Production, but Decreased LPS-Induced IL-10 Production in AM and Mo

As shown previously, LPS stimulation induced IL-12 p40 expression in both AM and Mo without inducing IL-12 p70 production. The addition of IFN- during LPS stimulation, however, induced IL-12 p70 and decreased IL-10 production. To determine the lowest concentration of IFN- able to synergize with LPS to upregulate IL-12 production and to decrease IL-10 production, AM and Mo were stimulated with a dose-response of IFN- (0 to 1,000 U/ml) and with a constant dose of LPS (10 µg/ml). As shown in Figure 3, in the absence of IFN-, LPS induced IL-12 p40 and IL-10 production but not IL-12 p70 production in both AM and Mo. The addition of IFN- (10-1,000 U/ml) during LPS stimulation induced a significant dose-dependent increase of IL-12 p40 and IL-12 p70 production and a dose-dependent inhibition of IL-10 production in both AM and Mo (P values < 0.05 by paired Student's t test). The highest dose of IFN- (1,000 U/ml) increased IL-12 p40 production by 2- to 5-fold in LPS-stimulated AM and Mo, induced IL-12 p70 production, and strongly reduced IL-10 production by 70 ± 8% and 55 ± 11%, respectively.

View larger version (17K): [in this window] [in a new window]   Figure 3.   IFN- increased LPS-induced IL-12 p40 and IL-12 p70 production and decreased LPS-induced IL-10 production in AM and Mo. AM and Mo (1 × 106 cells/ml) were stimulated by LPS (10 µg/ml) in the presence of increasing concentrations of IFN- (0, 0.1, 1, 10, 100, 1,000 U/ml). The production of IL-12 p40 (A), IL-12 p70 (B), and IL-10 (C) was measured in the culture supernatants by ELISA after 24 h of stimulation. Results are expressed as means ± SEM (n = 5). *P < 0.05 by paired Student's t test.

IL-10 Strongly Decreased LPS- and IFN--Induced IL-12 p40 and IL-12 p70 Production in AM

To determine which cytokine regulates the production of IL-12 in AM, cells were stimulated by LPS (10 µg/ml) and IFN- (1,000 U/ml) in the presence of rhIL-4, rhIL-10, rhTNF-, rhIL-1, and rhIL-1 (10 ng/ml). As shown in Table 1, IL-10 significantly decreased IL-12 p70 production by > 85% (P < 0.05) after 24 h of stimulation. In contrast to IL-10, the presence of IL-4, TNF-, IL-1, or IL-1 during LPS and IFN- stimulation did not modulate the production of IL-12 p70 by AM. When the cytokines were used alone, IL-12 p70 was not produced (data not shown).

The strong inhibitory effect of IL-10 on IL-12 production was further confirmed in a dose-dependent assay. AM were stimulated by LPS and IFN- in the presence of a dose response of rhIL-10 (0 to 5,000 pg/ml). As shown in Figure 4, IL-10 induced a dose-dependent decrease of both IL-12 p40 and IL-12 p70 production in AM. A significant inhibition of IL-12 p40 and IL-12 p70 production was observed with concentrations of IL-10 between 500 and 5,000 pg/ml. IL-12 p70 production was completely inhibited by the highest concentration of IL-10 (5,000 pg/ml), whereas IL-12 p40 production, although strongly reduced by IL-10 by ~ 85 ± 5%, was not abolished. This experiment further demonstrated the strong regulatory effect of IL-10 on IL-12 production by AM. IL-10 inhibition was already observed in the range of concentrations of IL-10, which has been measured in the culture supernatants of the previous experiments (Figure 3C).

View larger version (17K): [in this window] [in a new window]   Figure 4.   IL-10 inhibits IL-12 p40 and IL-12 p70 production in AM. AM (1 × 106 cells/ml) were stimulated by LPS (10 µg/ml) and IFN- (1,000 U/ml) in the presence of increased concentrations of rhIL-10 (0, 50, 100, 500, 1,000, 5,000 pg/ml). The production of IL-12 p40 (A) and IL-12 p70 (B) was measured in the culture supernatants by ELISA after 24 h of stimulation. Results are expressed as means ± SEM (n = 4). *P < 0.05 by paired Student's t test.

LPS- and IFN--Stimulated Mo Tend to Produce Higher Levels of IL-10 and Lower Levels of IL-12 p70 than AM

The capacity of AM and Mo to release both IL-10 and IL-12 p70 was compared. AM (n = 14) and Mo (n = 11) from unrelated donors were stimulated by LPS and IFN- as previously described, and the levels of IL-10 and IL-12 p70 were measured in the same supernatants after 24 h of stimulation. For each donor, the level of IL-12 p70 was reported on the x-axis, and the level of IL-10 was reported on the y-axis. As shown in Figure 5, three populations were clearly identified: (1) a group of cells constituted only by Mo (n = 6), which released high levels of IL-10 without detectable production of IL-12 p70; (2) a group constituted of both Mo (n = 5) and AM (n = 11), which released both IL-10 and IL-12 p70; and (3) a group of cells constituted only of AM (n = 3), which released high levels of IL-12 p70 with only traces of IL-10. Although no inverse correlation could be measured between the levels of IL-12 p70 and IL-10 induced in the same donors by LPS and IFN- stimulation, this experiment suggests that Mo tend to produce higher levels of IL-10 but lower levels of IL-12 p70 than AM.

View larger version (20K): [in this window] [in a new window]   Figure 5.   IL-12 p70 and IL-10 production by human AM and Mo. Human AM and Mo (1 × 106 cells/ml) were stimulated by LPS (10 µg/ml) and IFN- (1,000 U/ml). The production of IL-10 and IL-12 p70 was measured in the supernatants by ELISA after 24 h of stimulation. The data represent IL-10 and IL-12 p70 production released by AM (n = 14) and Mo (n = 11). Three populations of cells were characterized by their capacity to release (a) high levels of IL-10 and low levels of IL-12 p70, (b) high levels of both IL-10 and IL-12 p70, and (c) low levels of IL-10 and high levels of IL-12 p70.

Enhancement of LPS-Induced IL-12 p40 and IL-12 p70 Production in AM by Neutralization of Endogenously Produced IL-10

To investigate the autocrine regulation of IL-10 in IL-12 production by AM, cells were stimulated by LPS (10 µg/ml) in the presence or absence of neutralizing anti-IL-10 mAb (5 µg/ml). As shown in Figure 6, LPS induced high levels of IL-12 p40 without inducing IL-12 p70 production. The presence of anti-IL-10 mAb during LPS stimulation further increased IL-12 p40 production ~ twofold and induced a slight but significant production of IL-12 p70 (36 ± 14 pg/ml). Anti-IL-10 mAb alone did not induce either IL-12 p40 or IL-12 p70. Moreover, irrelevant control immunoglobulin G1 mAb did not further enhance LPS-induced IL-12 p70 production in any of three experiments (data not shown). These results exclude a direct role of anti-IL-10 antibody or of immune complexes in the induction of IL-12 production in AM. Therefore, neutralization of IL-10 production during LPS stimulation is sufficient to allow the production and release of IL-12 p40 and IL-12 p70 in AM in the absence of IFN-. Thus, the autocrine production of IL-10 by AM following LPS stimulation is sufficient to abrogate IL-12 production.

View larger version (24K): [in this window] [in a new window]   Figure 6.   Anti-IL-10 mAb increased the levels of LPS-induced IL-12 p40 and IL-12 p70 production by AM. AM (1 × 106 cells/ ml) were stimulated by LPS (10 µg/ml) in the absence or presence of anti-IL-10 mAb (5 µg/ml). The production of IL-12 p40 (A) and IL-12 p70 (B) was measured in the culture supernatants by ELISA after 24 h of stimulation. Results are expressed as means ± SEM (n = 6). *P < 0.05 by Wilcoxon signed rank test.

Enhancement of LPS- and IFN--Induced IL-12 p70 Production in AM by Neutralization of Endogenously Produced IL-10

The regulatory role of IL-10 in IL-12 production was further assessed in LPS- and IFN--stimulated AM. As shown in Figure 7, LPS and IFN- induced a strong production of IL-12 p70 (265 ± 86 pg/ml, n = 6) that was further enhanced two to threefold when anti-IL-10 mAb was added during LPS and IFN- stimulation (611 ± 160 pg/ml). Interestingly, in contrast to IL-12 p70, the addition of anti- IL-10 mAb during LPS and IFN- stimulation did not further enhance IL-12 p40 production, although IFN- increased the level of LPS-induced IL-12 p40 production by ~ threefold (Figure 6 compared with Figure 7).

View larger version (31K): [in this window] [in a new window]   Figure 7.   Anti-IL-10 mAb increased the levels of LPS- and IFN--induced IL-12 p70 production on AM. AM (1 × 106 cells/ ml) were stimulated by LPS (10 µg/ml) and IFN- (1,000 U/ml) in the absence or presence of anti-IL-10 mAb (5 µg/ml). The production of IL-12 p40 (A) and IL-12 p70 (B) was measured in the culture supernatants by ELISA after 24 h of stimulation. Results are expressed as means ± SEM (n = 6). *P < 0.05 by Wilcoxon signed rank test. ns = nonsignificant.

CD40-Stimulated AM Released IL-12 p40 and IL-12 p70 in the Absence of IL-10 Production

Several recent reports have described the capacity of human dendritic cells to release IL-12 following CD40 cross-linking. We therefore assessed the capacity of anti-CD40 mAb immobilized on CD32-transfected L cells to induce IL-10 and IL-12 production in AM. As shown in Figure 8, CD40 cross-linking alone failed to induce IL-12 p40 or IL-12 p70 production in AM. The addition of IFN- during CD40 cross-linking induced the production of both IL-12 p40 and IL-12 p70. CD40-induced IL-12 p40 and IL-12 p70 production, however, were ~ three to fourfold lower than that induced by LPS and IFN-. In contrast to LPS- and IFN--induced stimulation of IL-12 p70, the presence of anti-IL-10 mAb during CD40 and IFN- stimulation did not further enhance the production of either IL-12 p40 or IL-12 p70. When IL-10 production was measured in the same supernatants, we found that CD40 cross-linking induced trace amounts of IL-10 compared with stimulation by LPS and IFN-. Therefore, CD40 cross-linking in the presence of IFN- induces IL-12 p40 and IL-12 p70 production in AM in the absence of significant amounts of IL-10, although the level of both cytokines is lower than those produced by LPS and IFN-.

View larger version (23K): [in this window] [in a new window]   Figure 8.   Anti-CD40 mAb immobilized on CD32-transfected L cells induced IL-12 p40 and IL-12 p70 production without significant IL-10 production on AM. AM (1 × 106 cells/ml) were stimulated by CD32-transfected L cells (1 × 105 cells/ml) and anti-CD40 mAb (500 ng/ml) in the presence or absence of IFN- (1,000 U/ml) and anti-IL-10 mAb (5 µg/ml). As a control, AM were stimulated by LPS (10 µg/ml) and IFN- (1,000 U/ml). The production of IL-12 p40 (A), IL-12 p70 (B), and IL-10 (C) was measured in the culture supernatants by ELISA after 24 h of stimulation. Results are expressed as means ± SEM (n = 3). *P < 0.05 by paired Student's t test. ns = nonsignificant.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

In this report, we show that human AM produce lower levels of IL-12 p40 but similar and often higher levels of IL-12 p70 than do peripheral blood Mo when stimulated by LPS and IFN-. LPS-induced IL-12 p70 production was shown to be under the tight control of autocrine production of IL-10, whereas CD40-stimulated AM released IL-12 p70 with little or no concomitant production of IL-10.

The aim of this study was to compare the capacity of bacterial products (LPS) to induce IL-12 production in AM with CD40 cross-linking and to assess the regulatory role of IFN- and IL-10 on IL-12 production. Previous reports have shown that human Mo and dendritic cells released IL-12 when stimulated by LPS, or upon stimulation of CD40 molecules with CD40L expressed on activated T cells (5, 26). A recent report suggested that murine macrophages were able to produce IL-12 (29). However, no data are available regarding the capacity of human macrophages to produce IL-12.

Analysis of p35 and p40 mRNA subunits of IL-12 by RT-PCR showed that in unstimulated AM, p35 mRNA was slightly detectable, whereas p40 mRNAs was not expressed. The expression of both mRNAs was induced by LPS, and the presence of IFN- during LPS stimulation further increased p35 and p40 mRNA expression. Similar expression of p35 and p40 mRNA has been observed in peripheral blood Mo (9, 13), although both p35 and p40 mRNAs were decreased more rapidly in peripheral blood Mo than in AM.

AM cultured in medium alone did not release IL-12 p40, in contrast to Mo, in which spontaneous production of IL-12 p40 was detected. LPS induced IL-12 p40 production in AM and further increased its expression in Mo. The level of LPS-induced IL-12 p40 in AM was, however, 6- to 8-fold lower than that induced in Mo at any time of stimulation. IL-12 p70 production was not induced by LPS alone in AM or Mo. IFN- was a strong enhancer of LPS-induced IL-12 p40 production, confirming AM previous studies performed on Mo (9, 12, 13). IFN- strongly increased the capacity of LPS-stimulated AM and Mo to release IL-12 p70, which was induced more often at higher levels in AM than in Mo. The higher ratio of IL-12 p70 to IL-12 p40 production in AM than in Mo is of interest because secretion of a large excess of the free p40 chain has raised the question of whether p40 could compete for binding to IL-12 receptors with the biologically active IL-12 p70, and therefore act as a natural antagonist. Indeed, crude murine preparations of recombinant p40 were shown to antagonize the biologic activity of IL-12 (30). However, these results were partially confirmed in humans, in which only p40 homodimers, but not free p40 chains, have a modest inhibitory activity at a very high molar ratio (31). Therefore, although a physiologic role of p40 homodimers as natural antagonists remains to be demonstrated in humans, the higher susceptibility of IL-12 p70 as compared with IL-12 p40 to be upregulated by IFN- in AM and Mo demonstrates the crucial role of IFN- in favoring production of the biologically active heterodimer on these cells, particularly in AM.

LPS- and IFN--stimulated AM released not only IL-12 p40 and IL-12 p70 but also IL-10. In keeping with its general inhibitory properties, IL-10 is a strong inhibitor of IL-12 production (14, 15). We found that LPS-stimulated AM released high levels of IL-10 without producing IL-12 p70. The presence of IFN- during LPS stimulation, however, decreased the capacity of AM to release IL-10 and strongly enhanced both IL-12 p40 and IL-12 p70 production. The inhibitory effect of IFN- on IL-10 production suggests that the selective enhancer effect of IFN- on IL-12 p40 and IL-12 p70 production is at least partially mediated through an inhibition of IL-10 production. The efficiency of IL-10 to decrease IL-12 production by AM was confirmed by adding exogenous IL-10 to LPS- and IFN-- stimulated AM. IL-12 p70 production was markedly reduced by concentrations of IL-10 as low as 500 pg/ml and was almost completely abolished at 5,000 pg/ml. Meanwhile, IL-12 p40 production was reduced by 85 ± 5% with the highest concentration of IL-10. In contrast to IL-10, the presence of exogenous IL-1, IL-1, TNF-, or IL-4 did not significantly reduce the level of LPS- and IFN-- induced IL-12 p70 in AM. IL-4 and IL-10 are usually regarded as deactivating factors of Mo (32). Among the inhibitory cytokines, however, IL-4 appears to exhibit rather complicated effects on IL-12 production by either increasing or decreasing IL-12 p70 protein in human Mo, depending on the concentration that is used (15). In LPS- and IFN--stimulated AM, high concentrations of IL-4 did not significantly decrease the production of IL-12 p70. These experiments demonstrate that IL-12 production in AM is not regulated by IL-1, IL-4, or TNF-, but is strongly decreased by IL-10.

We further analyzed the inhibition mechanism of IL-12 p70 production by the autocrine production of IL-10 in AM. To estimate the autocrine role of IL-10 on IL-12 production, AM were stimulated by LPS alone, or by LPS and IFN-, in the presence of neutralizing anti-IL-10 mAb. Neutralization of autocrine production of IL-10 increased ~ twofold the level of LPS-induced IL-12 p40, and allowed the stimulation of IL-12 p70 by LPS in the absence of IFN-. These results confirm that IL-12 p40 and IL-12 p70 production on LPS-stimulated AM are tightly regulated by autocrine production of IL-10, and show that IFN- is not always required to induce IL-12 p70 production in these cells. IL-10 neutralization further increased the level of IL-12 p70 production in LPS- and IFN--stimulated AM but did not further increase the level of IL-12 p40. These experiments demonstrate the strong regulation of IL-12 p70 in AM by the autocrine production of IL-10 in the presence as well as in the absence of IFN-.

By using CD32-transfected L cells as a support for anti-CD40 mAb, we found that not only monocytes or dendritic cells (4, 5, 27), but also AM could be stimulated by CD40 cross-linking to release IL-12 p40 and IL-12 p70. The presence of IFN-, in addition to CD40 cross-linking, was needed in AM to induce IL-12 p40 and IL-12 p70 production. The levels of IL-12 p40 and IL-12 p70 produced by CD40 cross-linking were, however, lower than those induced by LPS and IFN-. This suggests that bacterial products are probably the primary mechanism of IL-12 induction in AM, whereas the interaction of dendritic cells (DC) with antigen-activated T cells has been postulated to be the primary mechanism of IL-12 production by DC. Thus, in both AM and DC, IL-12 production can be stimulated by a pathogen-induced T-cell-independent mechanism and by a separate T-cell-dependent mechanism that depends on a CD40L-CD40 interaction (33). Interestingly, CD40-stimulated AM released IL-12 without significant production of IL-10. The addition of anti-IL-10 mAbs did not increase CD40-induced IL-12 p70 production, in contrast to LPS-induced IL-12 p70 production. This suggests that AM may regulate their own production of IL-12 by autocrine production of IL-10 only when stimulated by bacterial products and when T cells are absent, but probably not when they are stimulated in a T-cell-dependent mechanism. The recent discovery that IL-12 induces IL-10 production by human T cells (16) raises the hypothesis of a negative feedback mechanism by T cells for IL-12- induced immune responses by monocytes/macrophages.

Our data provide strong evidence for the ability of IFN- to increase IL-12 production substantially in AM, with the requirement of a primary signal, provided in this study not only by LPS but also by CD40 cross-linking. The finding that production of bioactive IL-12 by accessory cells such as AM is regulated by T cells through CD40L- CD40 interaction and IFN- production implies that activated T cells may affect, by cell-cell contact mechanisms and by their secreted products, the cytokine profile of the accessory cells in their environment. Notably, both IL-12 and IFN- are involved in a positive feedback loop in which each enhances the production of the other (34), whereas IL-10 production by either AM or T cells inhibits it (14, 15). These regulatory mechanisms are likely to be of importance in human lungs, where not only bacterial products, but also activated T cells, may be in close contact with pulmonary macrophages. Depending on the type of activated T cells in close proximity of AM, this interaction may then result not only in potentiation of immune response by enhancing IL-12 production, but also in downregulation of this response by IL-10 released by T cells (14).

In this study, we have provided evidence that IL-12 production by AM in human lung may be strongly induced by bacterial products such as LPS and tightly controlled by LPS-induced IL-10 production. IFN- provided additional signals, beyond that of LPS, to enhance IL-12 p70 production and strongly decrease IL-10 production. We showed that AM could be activated by CD40-cross-linking to release IL-12 p70 without significant concomitant production of IL-10. In the latter condition, IL-12 p70 production by AM strongly depends on the relative amount of IFN- and IL-10 provided by activated T cells alone, which will either increase or inhibit its production.