In our study of the immunoregulatory roles of IL-10 in innate immunity, nonantigenic phagocytosable chitin particles were administered i.v. to IL-10-deficient (knockout (KO)) mice or KO mice pretreated with anti-NK1.1 or anti-IFN-γ Abs. The results established that chitin treatment of KO mice increased superoxide anion release from alveolar macrophages (Mφ) to a level much higher than that in wild-type (WT) mice. The results also suggested that the NK cell is the source of IFN-γ that is primarily responsible for this alveolar Mφ priming. To further study the roles of IL-10-inhibiting chitin-induced IFN-γ production, we used spleen cell cultures. The experiments showed that IL-12, IL-18, and TNF-α, which were produced by chitin-stimulated Mφ, contributed to the IFN-γ-inducing activity of chitin. Our results established that exogenous IL-10 inhibited chitin-induced IFN-γ production in spleen cell cultures from both KO and WT mice. Exogenous IL-10 also inhibited IL-12 and TNF-α production by chitin-stimulated Mφ. Exogenous IL-10 decreased IL-12- or IL-18-induced IFN-γ levels in KO but not in WT NK cell cultures. However, exogenous IL-10 enhanced IFN-γ levels when NK cells were stimulated simultaneously with both IL-12 and IL-18 in KO and WT cultures. Our in vitro data indicate that IL-10 has differential effects on chitin-induced IFN-γ production. However, the inhibitory effects of endogenous IL-10 appear to be dominant in the chitin-induced alveolar Mφ priming response in vivo.

Several bacterial components that induce innate immunity have been identified. LPS, exotoxin superantigens, and bacterial DNA are able to induce IL-12/TNF-α/IL-18 production by Mφ,3 all of which are extracellular signaling cytokines for IFN-γ production (1, 2, 3, 4). IL-18, an 18-kDa IFN-γ-inducing factor, acts synergistically with IL-12 to induce IFN-γ production by Th1 cells (4).

We recently found that phagocytosable chitin (N-acetyl-d-glucosamine polymer) is another inducer of innate immunity (5, 6). Our studies (5, 6) showed that: 1) splenic Mφ phagocytose chitin through mannose receptors to produce IL-12/TNF-α; 2) unlike LPS-induced cytokine production, the mechanism of this phagocytosis-induced cytokine production involves cytochalasin D-sensitive actin polymerization; 3) the Mφ-derived cytokines stimulate NK cells to produce IFN-γ; and 4) this IFN-γ production is negatively regulated by IL-4, TGF-β, PGE2, and IL-10. When C57BL/6 and SCID mice received chitin i.v., alveolar Mφ were activated within 3 days to become bactericidal. We further demonstrated that endogenous IFN-γ produced by NK cells is responsible for this alveolar Mφ priming (6).

IL-10 is an important negative regulator of cell-mediated immunity/Th1 functions (7). IL-10 is secreted by several different cell populations including T helper cells (Th1/Th2/Th0), monocytes, Mφ, B cells, and keratinocytes (7). IL-10 has been characterized as a factor that inhibits IFN-γ secretion from activated Th1 lymphocytes and NK cells (8, 9). In vitro studies have shown that IL-10 suppresses production of cytokines (IL-1, IL-6, TNF-α, GM-CSF, IL-12), generation of a reactive oxygen intermediate, and expression of surface MHC class II and costimulatory molecules such as B7 (10, 11, 12, 13). The immunoregulatory roles of IL-10 in vivo, however, appear to be complex. Dai et al. (14) have recently demonstrated that IL-10-deficient mice are resistant to Listeria monocytogenes infections due to high levels of endogenous IFN-γ production during the infection. Murray et al. (15) have shown that IL-10-transgenic mice are unable to clear mycobacterial infection. Surprisingly, excess IL-10 does not inhibit T cell responses to mycobacteria and IFN-γ production in these mice (15).

This study in which IL-10-deficient (KO) mice are used was to define the regulatory role of IL-10 with respect to: 1) the priming of alveolar Mφ by chitin in vivo; 2) the production of IFN-γ-inducing factors (IL-12, IL-18, TNF-α) by chitin-stimulated splenic Mφ in vitro; and 3) the production of IFN-γ by NK cells stimulated by these Mφ-derived cytokine(s) in vitro.

Breeding pairs of IL-10-deficient mice (C57BL/6-Il10tm1Cgn) (16) were obtained from The Jackson Laboratory (Bar Harbor, ME). Offspring were raised under pathogen-free conditions. Nonpregnant females, 8 to 14 wk old, were used for experiments. Age-matched female C57BL/6 mice were obtained from The Jackson Laboratory and used as wild-type control (WT) mice. Both IL-10-deficient (KO) and WT mice were maintained in barrier-filtered cages and fed Purina laboratory chow and tap water ad libitum.

Chitin particles (1–10 μm in diameter) were prepared from chitin powders (Sigma Chemical, St. Louis, MO) as described previously (5), suspended in saline (1 mg/ml), autoclaved, and kept at 4°C until use. Chitin preparations contained undetectable levels of endotoxin (<0.03 EU/ml). Cultured bacteria of Mycobacterium bovis Calmette-Guérin bacillus (BCG) Tokyo 172 strain were washed, autoclaved, and lyophilized. The powder of heat-killed (HK)-BCG was suspended in saline immediately before use. The suspensions of both chitin and HK-BCG were dispersed by brief sonication (10 s) when used. LPS (Escherichia coli 0111:B4, phenol) was obtained from Sigma.

To prime alveolar Mφ in vivo, mice were injected i.v. with 0.2 mg of chitin suspended in 0.2 ml of endotoxin-free saline. The alveolar Mφ were harvested 1 to 7 days after injection (5).

Endogenously produced IFN-γ was neutralized by injecting mice i.p. with 2 × 105 neutralizing units of anti-IFN-γ mAb (R4-6A2; specific activity, 2 × 105 neutralizing units per mg of IgG) 1 day before the chitin injection (5). An equivalent amount of normal rat IgG (Sigma) was used to control for the nonspecific effects of injecting foreign Ig.

As described previously (5), mice received i.p. 5 mg of purified anti-NK1.1 (IgG2a; clone PK136 from the American Type Culture Collection (ATCC), Manassas, VA) 1 day before chitin administration.

Alveolar Mφ were obtained by five repetitions of bronchopulmonary lavage with 1 ml of sterile HBSS, pH 7.2. Mφ enrichment was performed by the plastic adherence method (37°C, 1 h) in the presence of 10% heat-inactivated FBS. To prepare spleen cells, at least four spleens were pooled for each in vitro experiment. Plastic-adherent spleen Mφ were prepared as described previously (17). In indicated experiments, NK cell-enriched spleen cell populations were prepared as follows: CD4+ cells, CD8+ cells, Ly-6G (Gr-1)+ cells, B220 (CD45R)+ cells in the spleen cells were eliminated with a mixture of mAbs against CD4 (clone GK1.5 from ATCC), CD8 (clone 2.43; purified mAb was a gift from M. Evans, East Carolina University School of Medicine, Greenville, NC); Ly-6G (clone RB6-8C5 was a gift from R. L. Coffman, DNAX), and B220 (clone RA3-6B2; purified mAb was a gift from M. Evans), followed by treatment with rabbit serum (1:10, Sigma) as a source of complement. Adherent Mφ and damaged cells were removed by passage through a Sephadex G-10 column (18). The expression of surface antigens (Mac-1, NK1.1) on the Mφ and NK cell preparations, respectively, was determined by indirect immunofluorescence in the presence of 5% heat-inactivated newborn calf serum (Life Technologies, Grand Island, NY), pH 7.2, as described previously (5). Nucleated cell numbers, cell viability, and differential cell counts in freshly isolated cells and cultured cells were performed as described previously (5).

Superoxide dismutase (SOD)-inhibitable superoxide anion levels released by alveolar Mφ were measured by a cytochrome c reduction assay as described previously (5). Briefly, alveolar lavage cells were placed in a 24-well plate (Corning, Corning, NY) with HEPES-bicarbonate buffer containing 50 μM ferricytochrome c (Sigma). The adherent cells (>95% Mφ, determined by morphology) were incubated at 37°C for 1 h in the presence of PMA (1 μM). SOD (700 U/ml; Sigma) was also added as a negative control. The amount of reduced ferricytochrome c was measured by using a molecular extinction coefficient of 21.1 mM−1 cm−1 from the change in absorbance at 550 nm against a cell-free blank. Superoxide formation was expressed as nanomols per 106 cells.

Spleen cells (4 × 106 cells/ml) or plastic-adherent Mφ (106 cells/ml) in RPMI 1640 plus 10% heat-inactivated FBS were incubated with chitin at 100 μg/ml, HK-BCG at 100 μg/ml, or LPS at 100 ng/ml at 37°C. After 24 h of incubation, the culture supernatants were harvested, filtered through a 0.22-μm pore size Zetapore filter (Cuno, Meriden, CT) which removes particles and endotoxin, and stored at −80°C for later assays for cytokines. In some experiments, chitin particle-stimulated spleen cell cultures were further treated with recombinant mouse IL-10 (Pepro Tech, Rocky Hill, NJ) or Abs (rat anti-mouse IL-12 (clone 17.8; Genzyme, Cambridge, MA), polyclonal rabbit anti-mouse TNF-α (Genzyme), or polyclonal rabbit anti-mouse IL-18 (4). IFN-γ production was also performed using NK cell-enriched spleen cells (106 cells/ml) which were incubated with exogenous IL-12 (Genzyme) and/or IL-18 (4) at various doses or with the chitin-stimulated Mφ culture filtrates described above. After 24 h of incubation at 37°C, the supernatants were collected, and IFN-γ levels in the supernatants were measured by ELISA (5). The levels of IL-12p70 and IL-10 were also determined by specific ELISA (6). TNF-α bioactivity was measured by its cytotoxicity for L929 fibroblasts as described previously (6).

Differences between mean values were analyzed by Student’s t test. p values of <0.05 were considered statistically significant.

Chitin particle suspensions were given i.v. to KO and WT mice (1 mg/ml, 0.2 ml/injection). After selected periods, superoxide anion release by PMA-elicited alveolar Mφ was detected as described in Materials and Methods. PMA-elicited alveolar Mφ in WT mice generated <0.2 nmol/106 cells/h superoxide anion. Superoxide anion release was greatly enhanced 1 to 3 days after the injection of chitin and returned to normal (baseline) by day 7, which was consistent with previous reports (5). The kinetics of chitin-induced superoxide anion release in KO mice was similar to that of WT mice, peaking at day 3 and returning to normal by day 7. However, the magnitude of superoxide anion release was significantly increased compared with that of WT mice throughout the entire 7 days (Fig. 1). SOD at 700 U/ml completely inhibited superoxide anion release by alveolar Mφ (data not shown).

FIGURE 1.

Kinetics of superoxide anion release by alveolar Mφ in KO mice given chitin i.v. KO (▪) and WT (□) mice received 0.2 mg of chitin i.v. (3 mice/group). After the numbers of days indicated, alveolar Mφ in each mouse were assayed in vitro for superoxide anion release by PMA (1 μM). Results are expressed as mean ± SD, n = 3. ∗∗, p < 0.01 and ∗∗∗, p < 0.001 compared with WT mice.

FIGURE 1.

Kinetics of superoxide anion release by alveolar Mφ in KO mice given chitin i.v. KO (▪) and WT (□) mice received 0.2 mg of chitin i.v. (3 mice/group). After the numbers of days indicated, alveolar Mφ in each mouse were assayed in vitro for superoxide anion release by PMA (1 μM). Results are expressed as mean ± SD, n = 3. ∗∗, p < 0.01 and ∗∗∗, p < 0.001 compared with WT mice.

Close modal

To determine whether endogenous IFN-γ and NK cells were responsible for the chitin-induced alveolar Mφ priming, we treated KO mice with rat anti-IFN-γ mAb or mouse anti-NK1.1 mAb 1 day before inoculating them with chitin. As shown previously (5), both treatments significantly decreased alveolar Mφ priming in KO mice as well as in WT mice (Fig. 2).

FIGURE 2.

In vivo priming of mouse alveolar Mφ induced by i.v. injection of chitin in KO mice pretreated with mAbs against IFN-γ or NK1.1. KO (▪) and WT (□) mice (three mice/group) were injected i.p. with rat anti-IFN-γ IgG at 1 mg (A) or mouse anti-NK1.1 IgG at 5 mg (B) 1 day before i.v. injection of chitin at 0.2 mg/mouse. Control mice in A and B received rat IgG (1 mg) or saline, respectively, before the injection of chitin. Three days after chitin injection, alveolar Mφ in each mouse were assayed in vitro for PMA-elicited superoxide anion release. Results are expressed as mean ± SD, n = 3. ∗, p < 0.05 and ∗∗, p < 0.01 compared with the control (rat Ig) or (saline) group.

FIGURE 2.

In vivo priming of mouse alveolar Mφ induced by i.v. injection of chitin in KO mice pretreated with mAbs against IFN-γ or NK1.1. KO (▪) and WT (□) mice (three mice/group) were injected i.p. with rat anti-IFN-γ IgG at 1 mg (A) or mouse anti-NK1.1 IgG at 5 mg (B) 1 day before i.v. injection of chitin at 0.2 mg/mouse. Control mice in A and B received rat IgG (1 mg) or saline, respectively, before the injection of chitin. Three days after chitin injection, alveolar Mφ in each mouse were assayed in vitro for PMA-elicited superoxide anion release. Results are expressed as mean ± SD, n = 3. ∗, p < 0.05 and ∗∗, p < 0.01 compared with the control (rat Ig) or (saline) group.

Close modal

Previously, we demonstrated that chitin induced production of IL-12p70 (bioactive IL-12), TNF-α, and IFN-γ in spleen cell cultures prepared from C57BL/6 mice (6). To define the role of IL-10, spleen cells isolated from KO mice were incubated with chitin particles. As shown in Table I, the levels of IL-12, TNF-α, and IFN-γ were markedly higher than those from WT mice. To determine whether these endogenous cytokines contributed IFN-γ production, spleen cell cultures were treated with neutralizing Abs against IL-12 and TNF-α before the stimulation of chitin. Abs against IL-12 and TNF-α inhibited the IFN-γ-inducing activities (Fig. 3). Anti-IL-18 also inhibited the IFN-γ-inducing activity (Fig. 3). When exogenous IL-10 was added to the spleen cell cultures, the levels of IFN-γ were dramatically reduced (Fig. 4). Treatments with IL-10 at 1 and 10 ng/ml resulted in >95% inhibition of the cytokine production.

Table I.

IFN-γ, IL-12, and TNF-α levels induced by chitin in spleen cell cultures prepared from KO and WT micea

StimulationConcentration (μg/ml)IFN-γ (U/ml)IL-12 (ng/ml)TNF-α (U/ml)
KO spleen cells stimulated with     
Medium  <1 <0.1 <0.1 
Chitin 100 450** 3.2** 34* 
HK-BCG 100 320** 2.4** 21* 
LPS 0.1 470** 3.7** 37* 
WT spleen cells stimulated with     
Medium  <1 <0.1 <0.1 
Chitin 100 58 0.6 12 
HK-BCG 100 46 0.5 11 
LPS 0.1 66 0.8 17 
StimulationConcentration (μg/ml)IFN-γ (U/ml)IL-12 (ng/ml)TNF-α (U/ml)
KO spleen cells stimulated with     
Medium  <1 <0.1 <0.1 
Chitin 100 450** 3.2** 34* 
HK-BCG 100 320** 2.4** 21* 
LPS 0.1 470** 3.7** 37* 
WT spleen cells stimulated with     
Medium  <1 <0.1 <0.1 
Chitin 100 58 0.6 12 
HK-BCG 100 46 0.5 11 
LPS 0.1 66 0.8 17 
a

Spleen cells from KO mice and WT mice were incubated with chitin, HK-BCG, and LPS at indicated concentrations for 24 h at 37°C. The levels of the cytokines in the culture supernatants were measured as described in Materials and Methods. Data represent the mean from triplicate samples. ∗, p < 0.05 and ∗∗, p < 0.01 compared with the WT control groups.

FIGURE 3.

Effects of neutralizing Abs against IL-12p70, TNF-α, and IL-18 on chitin-induced IFN-γ production. Spleen cells isolated from KO (▪) and WT (□) mice were incubated with chitin at 100 μg/ml in the presence of rat anti-IL-12 (40 μg/ml, clone 1.78), polyclonal rabbit anti-mouse TNF-α (40 μg/ml), or polyclonal rabbit anti-mouse IL-18 (50 μg/ml) for 24 h at 37°C. The control group had a mixture of rat Ig (40 μg/ml; Sigma) and rabbit Ig (50 μg/ml; Sigma). The levels of IFN-γ in the culture supernatants were measured as described in Materials and Methods. Results are expressed as mean ± SD from triplicate samples. ∗, p < 0.05, ∗∗, p < 0.01 and ∗∗∗, p < 0.001 compared with the control (Igs) groups.

FIGURE 3.

Effects of neutralizing Abs against IL-12p70, TNF-α, and IL-18 on chitin-induced IFN-γ production. Spleen cells isolated from KO (▪) and WT (□) mice were incubated with chitin at 100 μg/ml in the presence of rat anti-IL-12 (40 μg/ml, clone 1.78), polyclonal rabbit anti-mouse TNF-α (40 μg/ml), or polyclonal rabbit anti-mouse IL-18 (50 μg/ml) for 24 h at 37°C. The control group had a mixture of rat Ig (40 μg/ml; Sigma) and rabbit Ig (50 μg/ml; Sigma). The levels of IFN-γ in the culture supernatants were measured as described in Materials and Methods. Results are expressed as mean ± SD from triplicate samples. ∗, p < 0.05, ∗∗, p < 0.01 and ∗∗∗, p < 0.001 compared with the control (Igs) groups.

Close modal
FIGURE 4.

Inhibitory effects of exogenous IL-10 on chitin-induced IFN-γ production. KO and WT spleen cells were incubated with chitin in the presence of exogenous IL-10 (0.1, 1, and 10 ng/ml) for 24 h at 37°C. As comparison controls, the spleen cell cultures were stimulated with HK-BCG (100 μg/ml) or LPS (100 ng/ml) instead of chitin. The levels of IFN-γ in the culture supernatants were measured as described in Materials and Methods. Results are expressed as mean ± SD from triplicate samples.

FIGURE 4.

Inhibitory effects of exogenous IL-10 on chitin-induced IFN-γ production. KO and WT spleen cells were incubated with chitin in the presence of exogenous IL-10 (0.1, 1, and 10 ng/ml) for 24 h at 37°C. As comparison controls, the spleen cell cultures were stimulated with HK-BCG (100 μg/ml) or LPS (100 ng/ml) instead of chitin. The levels of IFN-γ in the culture supernatants were measured as described in Materials and Methods. Results are expressed as mean ± SD from triplicate samples.

Close modal

Several bacterial components including LPS initiate innate immunity with induction of IL-12 production (1). Our previous study showed that chitin and LPS stimulate IL-12 production by different mechanisms (6). In this comparison study, KO spleen cells were stimulated with LPS and HK-BCG. Like chitin, both HK-BCG and LPS increased IL-12, TNF-α, and IFN-γ production (Table I). Exogenous IL-10 added to the cultures markedly inhibited IFN-γ production (Fig. 4).

These results indicate that IL-10 down-regulates not only chitin-induced IFN-γ production but also BCG- or LPS-induced IFN-γ production. However, it is still unclear which step(s) of innate immunity induced by chitin is inhibited by IL-10.

Spleen cells isolated from IL-10-deficient mice and controls contained 6 and 7% of Mac-1+ cells, respectively (mean, n = 3, data not shown). Plastic-adherent Mφ preparations from KO and WT mice contained 70 and 74% Mac-1+ cells, respectively (data not shown). Splenic Mφ were stimulated with chitin in the presence or absence of exogenous IL-10. The levels of IL-12 and TNF-α produced in the Mφ cultures are shown in Table II. These results showed that when stimulated by chitin, KO Mφ produced six- and threefold more IL-12 and TNF-α, respectively, than WT Mφ. Treatment with exogenous IL-10 at 1 and 10 ng/ml resulted in >90% inhibition of the production of IL-12 and TNF-α (Table II).

Table II.

Inhibitory effects of IL-10 on the IL-12 and TNF-α production by splenic Mφa

Cytokine DetectedExogenous IL-10 (ng/ml)
0 (medium)0.1110
From chitin-stimulated KO Mφ     
IL-12p70 (ng/ml) 8.3 0.5** <0.1** <0.1** 
TNF-α (U/ml) 40 12* 0.3** 0.1** 
From chitin-stimulated WT Mφ     
IL-12p70 (ng/ml) 1.2 0.3** <0.1** <0.1** 
TNF-α (U/ml) 13 4* 0.2** <0.1** 
Cytokine DetectedExogenous IL-10 (ng/ml)
0 (medium)0.1110
From chitin-stimulated KO Mφ     
IL-12p70 (ng/ml) 8.3 0.5** <0.1** <0.1** 
TNF-α (U/ml) 40 12* 0.3** 0.1** 
From chitin-stimulated WT Mφ     
IL-12p70 (ng/ml) 1.2 0.3** <0.1** <0.1** 
TNF-α (U/ml) 13 4* 0.2** <0.1** 
a

Plastic-adherent Mφ isolated from KO and WT mice were incubated with chitin at 100 μg/ml for 24 h at 37°C. The levels of the cytokines in the culture filtrates were measured as described in Materials and Methods. Data represent the mean from triplicate samples. ∗, p < 0.05 and ∗∗, p < 0.01 compared with the medium control groups.

Splenic NK (NK1.1+) cells, but not CD4+ cells, are the major producers of IFN-γ in chitin-induced innate immunity (5). NK1.1+ cells accounted for 7% of the spleen cells in both KO and WT mice (data not shown). Following negative selection as described in Materials and Methods, NK1.1+ cells accounted for 67 and 71% from KO and WT mice, respectively (data not shown). To assess the effect of IL-10, NK cells were cultured with IL-12 and/or IL-18 for 24 h at 37°C. NK cells were also cultured with the chitin-stimulated KO Mφ culture filtrates prepared above.

WT NK cells responded to either IL-12 or IL-18 (both at 0.01–10 ng/ml) and released IFN-γ in a small but notable dose-dependent manner. Typical results at 1 ng/ml of the cytokines are shown in Table III. The combination of IL-12 and IL-18 showed synergistic effects on IFN-γ production (Table III). Similarly, the culture filtrates from chitin-, BCG-, and LPS-stimulated KO Mφ cultures (Table II; Fig. 3) induced IFN-γ production. The amounts of IFN-γ were significantly higher than those induced by IL-12 alone or IL-18 alone (Table III). When KO NK cells were stimulated with IL-12 alone or IL-18 alone, they released more IFN-γ than WT NK cells (Table III). In addition, neither synergistic nor additive effects of these two cytokines were observed. The levels of IFN-γ production induced by the mixtures of two cytokines were comparable to, but slightly lower than, those shown by WT NK cells (Table III).

Table III.

Effects of endogenous and exogenous IL-10 on IFN-γ production by splenic NK cellsa

Stimulation (Concentration)Exogenous IL-10 (ng/ml) Treatment
00.1110
KO NK cells stimulated with IFN-γ production (U/ml)    
Medium <1 <1 <1 <1 
IL-12 (1 ng/ml) 250 206 147* 142* 
IL-18 (1 ng/ml) 336 248* 184* 166* 
IL-12/IL-18 (1 ng/ml each) 370 391 480* 477* 
KO Mφ culture filtrates (50%) induced by     
Chitin 276 354* 410* 439* 
BCG 197 247 340* 354* 
LPS 268 311 385* 352* 
Medium <1 <1 <1 <1 
WT NK cells stimulated with     
Medium <1 <1 <1 <1 
IL-12 (1 ng/ml) 75 76 70 68 
IL-18 (1 ng/ml) 64 60 55 57 
IL-12/IL-18 (1 ng/ml each) 478 450 523 581* 
KO Mφ culture filtrates (50%) induced by     
Chitin 372 428 490* 473* 
BCG 325 333 360 418* 
LPS 374 380 475* 446* 
Medium <1 <1 <1 <1 
Stimulation (Concentration)Exogenous IL-10 (ng/ml) Treatment
00.1110
KO NK cells stimulated with IFN-γ production (U/ml)    
Medium <1 <1 <1 <1 
IL-12 (1 ng/ml) 250 206 147* 142* 
IL-18 (1 ng/ml) 336 248* 184* 166* 
IL-12/IL-18 (1 ng/ml each) 370 391 480* 477* 
KO Mφ culture filtrates (50%) induced by     
Chitin 276 354* 410* 439* 
BCG 197 247 340* 354* 
LPS 268 311 385* 352* 
Medium <1 <1 <1 <1 
WT NK cells stimulated with     
Medium <1 <1 <1 <1 
IL-12 (1 ng/ml) 75 76 70 68 
IL-18 (1 ng/ml) 64 60 55 57 
IL-12/IL-18 (1 ng/ml each) 478 450 523 581* 
KO Mφ culture filtrates (50%) induced by     
Chitin 372 428 490* 473* 
BCG 325 333 360 418* 
LPS 374 380 475* 446* 
Medium <1 <1 <1 <1 
a

NK cell-enriched spleen cells were isolated from KO and WT mice as described in Materials and Methods and were incubated with IL-12, IL-18, their mixtures, and KO Mφ culture filtrates prepared above (see Table II) at indicated doses. The KO Mφ culture filtrates, which were prepared from the cultures stimulated with chitin, BCG and LPS for 24 h, were described in Table II. These KO Mφ culture filtrates were added to the NK cell cultures at 1/1 dilution. Data represent the mean from triplicate samples. ∗, p < 0.05 compared with the groups treated with no IL-10.

To further assess the role of IL-10 regulating IFN-γ production, exogenous IL-10 was added to NK cell cultures described above. In KO NK cells, the levels of IL-12- or IL-18-induced IFN-γ production were decreased by exogenous IL-10 in a dose-dependent manner (Table III). Surprisingly, IFN-γ levels induced by the mixtures of IL-12/IL-18 or the chitin-stimulated KO Mφ culture filtrates were slightly but significantly increased (Table III). In contrast, the levels of IFN-γ production in WT mice, when stimulated by IL-12 alone or IL-18 alone, were unchanged by the exogenous IL-10 treatments (Table III). Exogenous IL-10 at 1 and 10 ng/ml, however, enhanced IFN-γ production at 20 to 40% when induced by the mixtures of IL-12/IL-18 or by the chitin-stimulated KO Mφ culture filtrates (Table III).

We measured the levels of IL-10 in the cultures of splenic Mφ and NK cells isolated from WT mice. As shown in Table IV, when Mφ were stimulated with chitin, 317 pg/ml IL-10 were detected. Comparable levels of IL-10 were also detected by stimulation with HK-BCG and LPS. Although neither IL-12 nor IL-18 induced detectable IL-10, the combination of these cytokines or the chitin-induced KO Mφ culture filtrates induced significant amounts of IL-10 production in NK cell cultures (Table IV). These results suggest that endogenous IL-10 produced in the chitin-induced innate immunity is derived, at least in part, from Mφ and NK cells.

Table IV.

IL-10 production in vitro by splenic Mφ and splenic NK cells isolated from WT micea

StimulationConcentrationIL-10 (pg/ml)
Mφ stimulated with   
Medium  <15 
Chitin 100 μg/ml 317 ± 29 
HK-BCG 100 μg/ml 370 ± 52 
LPS 0.1 μg/ml 68 ± 28 
NK cells stimulated with   
Medium  <15 
IL-12 10 ng/ml <15 
IL-18 10 ng/ml <15 
IL-12/IL-18 10 ng/ml each 108 ± 11 
IL-12/IL-18 1 ng/ml each 45 ± 7 
IL-12/IL-18 0.1 ng/ml each 32 ± 2 
KO Mφ culture filtrate (medium) 50% <15 
KO Mφ culture filtrate (chitin) 50% 85 ± 15 
StimulationConcentrationIL-10 (pg/ml)
Mφ stimulated with   
Medium  <15 
Chitin 100 μg/ml 317 ± 29 
HK-BCG 100 μg/ml 370 ± 52 
LPS 0.1 μg/ml 68 ± 28 
NK cells stimulated with   
Medium  <15 
IL-12 10 ng/ml <15 
IL-18 10 ng/ml <15 
IL-12/IL-18 10 ng/ml each 108 ± 11 
IL-12/IL-18 1 ng/ml each 45 ± 7 
IL-12/IL-18 0.1 ng/ml each 32 ± 2 
KO Mφ culture filtrate (medium) 50% <15 
KO Mφ culture filtrate (chitin) 50% 85 ± 15 
a

The culture conditions of splenic Mφ and NK cells from WT mice were described in Tables II and III, respectively. The KO Mφ culture filtrates, which were incubated with medium alone or chitin for 24 h, were prepared as described in Table II. These KO Mφ culture filtrates were added to the NK cell cultures at 1/1 dilution as described in Table III. IL-10 levels in the NK culture supernatants were determined by specific two-site ELISA with capture anti-IL-10 MAb (clone 16E3) and biotinylated anti-IL-10 MAb (clone 2A5) (both from Endogen, Woburn, MA). Results are expressed as mean ± SD from triplicate samples.

The striking features of the immunoregulatory roles of IL-10 described in this paper are as follows: 1) i.v. injection of chitin in KO mice increases superoxide anion release from alveolar Mφ as compared with WT mice (Fig. 1); 2) the mechanisms of NK cell-produced IFN-γ, which is primarily responsible for this alveolar Mφ priming, appear to be down-regulated by endogenous IL-10 (Fig. 2); 3) exogenous IL-10 inhibits chitin/BCG/LPS-induced IFN-γ production in both KO and WT spleen cell cultures (Fig. 4); 4) IL-12, TNF-α, and IL-18, which are produced by chitin-stimulated splenic Mφ (inhibition by IL-10), contribute to the IFN-γ-inducing activity of chitin (Fig. 3; Tables I and II); 5) exogenous IL-10 decreases IL-12- or IL-18-induced IFN-γ production by KO NK cells but not WT NK cells (Table III); and 6) exogenous IL-10, however, enhances IFN-γ levels when NK cells are stimulated simultaneously with both IL-12 and IL-18 in KO and WT cultures (Table III).

We have previously demonstrated that the mechanism of IL-12 and TNF-α production induced by chitin and HK-BCG involves mannose receptor-mediated phagocytosis that depends on cytochalasin D-sensitive actin polymerization events (6). In contrast, LPS-induced IL-12 production is independent of these cellular events (6). Based on the present study, however, it is clear that IL-10 modifies the Mφ responses not only to chitin and HK-BCG but also to LPS for the production of IL-12, TNF-α, and IL-18. We also found in this study that splenic Mφ produce IL-10 when stimulated with chitin/BCG/LPS (Table IV). Our results clearly indicate that consistent with earlier observations (10, 11, 12), IL-10 down-regulates the initial stages of innate immunity mediated by Mφ in an “autocrine” fashion.

IFN-γ production by NK cells is induced not only by IL-12 and TNF-α as previously described (3, 5) but also by IL-18 as described in this study. IL-18 produced by Mφ was originally identified and characterized as IFN-γ-inducing factor by Okamura et al. (4). Since then, synergistic effects between IL-12 and IL-18 on IFN-γ production by T-helper clones and CD40-stimulated B cell activation have been documented in other models (19, 20, 21). In the present study, IL-12 and IL-18 synergistically induce splenic NK cells to produce IFN-γ in WT mice. Such synergistic enhancement of IFN-γ production was initially observed by TNF-α and IL-12, although TNF-α alone does not induce IFN-γ production (9, 22). This mechanism can explain that the initial induction of IFN-γ by NK cells does not require detectable levels of IL-12 in chitin-induced innate immunity. This possibility is supported by our preliminary study where IL-12 at 1 pg/ml, undetectable by ELISA, does not induce IFN-γ production (>5 U/ml) by WT NK cells unless costimulated with IL-18 at 10 pg/ml (data not shown). In addition, previous studies including ours (1, 5, 22) indicate that endogenous IFN-γ has a stimulatory effect on Mφ and makes them more responsive to chitin.

A unique immunostimulatory effect of IL-10 on IFN-γ production is demonstrated in this study. IL-10 enhances IFN-γ levels when NK cells (KO or WT) are stimulated with chitin-stimulated KO Mφ culture filtrates containing both IL-12 and IL-18. Our additional results (Table III), however, suggest an exception that in some cases where Mφ produce either IL-12 or IL-18, IL-10 decreases IFN-γ production. Overall, our in vitro study indicates that IL-10 has differential effects on the chitin-induced innate immune responses: 1) inhibitory effect on Mφ cytokine production; and 2) stimulatory effect of IFN-γ production by NK cells. In the chitin-induced alveolar Mφ priming in vivo, the inhibitory effects of endogenous IL-10 appear to be dominant.

Murray et al. (15) found that overexpression of IL-10 does not inhibit but rather enhances IFN-γ secretion by purified protein derivative-stimulated spleen cells isolated from the BCG-immunized IL-10-transgenic mouse model. Although further confirmation of whether these observations are made particularly in the transgenic mice is required, this study strongly suggests that IL-10 directly enhances IFN-γ production by Ag-specific Th1 cells and/or nonspecifically by NK cells under chronic immunologic conditions. Furthermore, there are several studies indicating IL-10 as an immmunostimulator (23, 24, 25). For example, IL-10 injection in mice (200 μg/mouse/day) with expecting immunosuppressive roles in graft-vs-host diseases failed because IL-10 may have been an immunostimulant, which probably enhanced IFN-γ production (23).

Peritt et al. (26) documented that IL-12-induced NK cells produce IL-10. In this connection, it has been proposed that although both T cells and NK cells express IL-10 receptors, the effects of IL-10 on these two cells would differ (27, 28). Carson et al. (28), using human NK cells, found that: 1) IL-10 receptors are constitutively expressed on human NK cells; 2) unlike IL-2-activated T cells, the proliferation of IL-2-activated NK cells is further enhanced by IL-10; 3) the production of IFN-γ, TNF-α, and GM-CSF by IL-2-activated NK cells is significantly enhanced by IL-10; 4) IL-10 induces NK cell cytotoxic activity against tumor cells; and 5) IL-10 does not enhance IFN-γ production when human NK cells are stimulated by IL-12 alone. These observations and our results suggest that IL-10 inhibits or enhances NK cell functions in an “autocrine” fashion, depending on the cytokine milieu.

In addition to the inhibitory effects of IL-10 on IFN-γ production as described above, the following two additional mechanisms would be involved for the enhancement of chitin-induced alveolar Mφ priming in KO mice. First, it is well established that IL-10 inhibits the generation of a reactive oxygen intermediate and a reactive nitrogen intermediate by IFN-γ-induced effector Mφ (12, 29). This mechanism was further supported by our unpublished studies using an in vitro IFN-γ-induced alveolar Mφ priming assay (100 U/ml IFN-γ, 24 h) (5). We found that the capacities of PMA-elicited superoxide anion release are higher in IFN-γ-primed Mφ from KO mice than those from WT mice (5.6 and 3.8 nmol/106 cells/h, respectively, data not shown). Exogenous IL-10 at 1 to 100 ng/ml during the IFN-γ treatment inhibited superoxide anion release in a dose-dependent manner (up to 30% inhibition) in both KO and WT mice (data not shown). Unlike IL-4 (30), however, the inhibitory effect of IL-10 was not observed when exogenous IL-10 was added to alveolar Mφ, which had been primed previously either in vitro with IFN-γ or in vivo by chitin injection (data not shown). Secondly, the generation of the Mφ effector functions is induced by not only IFN-γ but also Mφ-derived cytokines including TNF-α (31), which is produced at higher levels by KO Mφ (Tables I and II).

The present study indicates major endogenous cytokines regulating innate immunity. The unique contribution of our series of studies (5, 6) is that nonantigenic and biodegradable chitin represents an effective tool to study immunoregulatory mechanisms of IL-10.

We thank Mark D. Mannie, East Carolina University School of Medicine, for his critical review of the manuscript. We also thank Mark Evans, East Carolina University, and Robert L. Coffman, DNAX, for their gifts of purified mAbs (anti-CD8 and anti-B220) and a hybridoma, RB6-8C5, respectively.

1

This work was supported by grants from the American Lung Association, the East Carolina University School of Medicine, North Carolina Biotechnology Center, and Pitt County Memorial Hospital Foundation.

3

Abbreviations used in this paper: Mφ, macrophages; HK, heat killed; SOD, superoxide dismutase; WT mice, wild-type control mice; KO mice, IL-10-deficient (knockout) mice; BCG, Calmette-Guérin bacillus.

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