We studied the potential role of a cytokine regulatory mechanism(s) in LPS-dependent reprogramming and modulation of TNF-α and nitric oxide (NO) responses in mouse peritoneal macrophages. Reciprocal regulation of TNF-α and NO production by LPS-primed and LPS-stimulated macrophages was found to be dependent on the presence of soluble secretory products released by the cells during the initial LPS priming interaction. Pretreatment of naïve macrophages with different mouse recombinant cytokines such as rIL-10, rIL-12, and rIFN-γ dose dependently and differentially regulated subsequent LPS-induced production of TNF-α, IL-6, and NO by cytokine-primed cells. Analysis of IL-12 and IL-10 levels present in culture supernatants of LPS-primed and LPS-stimulated macrophages revealed a high degree of correlation between the profiles of TNF-α and IL-12 as well as NO and IL-10. Furthermore, LPS priming of macrophages in the presence of anti-IL-12-neutralizing mAb attenuated TNF-α responses while at the same time up-regulated NO production. In contrast, neutralization of endogenous IL-10 with anti-IL-10 mAb resulted in considerable TNF-α response at LPS priming doses under conditions that would otherwise strongly inhibit TNF-α production. We also found that the initial LPS priming of naïve macrophages differentially and dose dependently regulates expression of mRNAs for IL-10, IL-12, and IFN-γ in LPS-primed macrophages. Collectively, our data provide experimental support for the hypothesis that a cytokine regulatory network, most probably autocrine, tightly controls the reciprocal modulation of TNF-α and NO responses in LPS-primed macrophages.
Endotoxin or LPS of Gram-negative bacteria is an integral component of the bacterial cell wall, and is known to mediate a number of biologic activities associated with the sepsis syndrome, including fever, hypotension, multiple organ failure, shock, and death (1). These deleterious host responses to endotoxin are believed to result from an uncontrolled cascade of proinflammatory cytokines produced by cells of the reticuloendothelial system in general, and macrophages in particular. Cytokines represent a family of intercellular mediators that are now recognized to play a central role in virtually all interactions involving cells of the immune system, e.g., communication and differentiation of cellular components of the immune system, induction and amplification of inflammatory responses, and generation of antiinflammatory reactions (2). Macrophages are considered to be one of the main cellular sources of cytokines released in the body in response to different microbial products, including LPS. In numerous in vitro studies, it has been demonstrated that LPS-dependent activation of macrophages results in the release of various secretory products, including proinflammatory cytokines such as TNF-α, IL-1, IL-6, IL-12, and antiinflammatory cytokines, e.g., IL-10. It is of importance that these cytokines can be both produced and utilized by macrophages in processes known as autocrine regulatory pathways.
A unique property of endotoxin is that it can modulate in mammals a transient state of either hypersensitivity to itself, seen in animals with bacterial infections (3), or low responsiveness induced by a single or repeated injections of low LPS amounts in human volunteers and experimental animals (reviewed in 4 . The later phenomenon, referred as endotoxin tolerance, and its molecular mechanism(s) has been the subject of extensive studies, in large part due to its potential application for correction and/or prevention of the pathophysiologic sequelae associated with endotoxemia and Gram-negative sepsis. Unlike the early interpretation of endotoxin tolerance as a protection from the lethal effects of bacterial pyrogens (5), the current concept of this phenomenon implies that LPS unresponsiveness is controlled at the cellular level, and that the activity of macrophages may well be instrumental in the development of LPS tolerance (6, 7, 8). Control of macrophage LPS responsiveness may be of primary importance for the host and may function to limit the extent of the proinflammatory response to protect the host from excessive destructive processes during inflammation and infection. However, it is becoming increasingly apparent that the acquisition of an endotoxin refractory state is not derived from complete unresponsiveness of exhausted macrophages exposed to chronic LPS stimulus. Rather, this process is mediated by a highly orchestrated compensatory mechanism(s) controlling the balance of pro- and antiinflammatory cytokines in the host (9, 10). In this respect, it is attractive to hypothesize that some microbial pathogens, and probably malignant cells, may utilize a similar strategy to create an imbalance in the coordinated proinflammatory response of the immune system in an effort to circumvent host immunity.
Although the phenomenon of modulation of LPS responsiveness was initially described in in vivo experiments, recent studies have demonstrated that in vitro pretreatment of naïve macrophages with LPS also results in modulation of a refractory state in macrophages to subsequent activation with LPS (11, 12, 13). Furthermore, we have recently demonstrated that in vitro pretreatment of macrophages with LPS may result in a more complex process than a simple abrogation of proinflammatory responses in the cells. In those studies, it was shown that pretreatment of elicited mouse peritoneal macrophages with different threshold LPS doses induces a complex of intracellular reprogramming events resulting in a differential dose-dependent secretory activity of macrophages upon restimulation with a secondary effective LPS dose (14, 15, 16). This reprogramming is characterized phenotypically by a biphasic enhancement/suppression of TNF-α responsiveness compared with a reciprocal suppression/enhancement of nitric oxide (NO)4 secretion. Although we have suggested that reprogramming of macrophages may represent a fundamental regulatory mechanism for governing the host responses to LPS and, perhaps, controlling immunity to infection in general, the exact biochemical and molecular mechanism(s) underlying LPS-induced priming events in macrophages has not been well defined.
The present study was undertaken to test experimentally our hypothesis that LPS-dependent priming of macrophages is mediated by a cytokine autocrine/paracrine regulatory network and, specifically, is dictated by a profile of pro- and antiinflammatory cytokines produced by the cells during the reprogramming stage.
Materials and Methods
C3Heb/FeJ mice of both sexes were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were maintained in the American Association for Accreditation of Laboratory Animal Care-certified Kansas University Medical Center animal facility under 12-h light/dark cycles with food and water provided ad libitum. Eight- to twelve-week-old mice were used for isolation of peritoneal macrophages.
Phenol-extracted LPS from Escherichia coli O111:B4 was purchased from List Biologic Laboratories (Campbell, CA). Mouse rIL-10 (sp. act. 5 × 105 U/mg) and mouse rIL-4 (sp. act. 107 U/mg) were obtained from PharMingen (San Diego, CA). Recombinant mouse IL-12 (sp. act. 5 × 105 U/mg) was purchased from Genzyme (Cambridge, MA). Recombinant mouse IFN-γ (sp. act. 1 × 106 U/mg) was a gift from Dr. S. W. Russell (University of Kansas Medical Center, Kansas City, KS). The endotoxin level in all recombinant cytokines used was less than 0.1 ng per μg of cytokine according to an analysis performed by the producer of the cytokines. Neutralizing rat anti-mouse IFN-γ mAb (IgG1; clone XMG1.2) was obtained from PharMingen, neutralizing rat anti-mouse IL-12 mAb (IgG2a; clone C17.8) was from Genzyme, and neutralizing rat anti-mouse IL-10 mAb (IgG1; clone JES5-2A5) was purchased from Biosource International (Camarillo, CA). According to the manufacturer’s analysis, endotoxin contamination of all mAbs utilized was less than 0.01 ng/μg of protein. Endotoxin-tested RPMI 1640 culture medium and heat-inactivated FBS (endotoxin content, less than 0.6 endotoxin U/ml) was purchased from Sigma (St. Louis, MO).
Isolation and culture of peritoneal macrophages
Macrophages were obtained from C3Heb/FeJ mice i.p. injected with 1.5 ml of 4% thioglycollate broth (Difco Laboratories, Detroit, MI) 4 days before cell isolation. Macrophages were harvested by peritoneal lavage with sterile HBSS and washed twice with the same solution by centrifugation at 800 × g for 10 min. Isolated cells were resuspended in serum-free RPMI 1640 culture medium supplemented with 100 U/ml of penicillin and 100 μg/ml of streptomycin, counted, and dispensed into either 24-well tissue culture plates (Costar, Cambridge, MA) or 6-well plates (Costar) at an approximate density of 1 × 106 and 5 × 106 cells per well, respectively. Cells were incubated in 5% CO2 humidified atmosphere of a CO2 incubator for 20 to 30 min at 37°C before nonadherent cells (primarily lymphocytes) were removed by washing with HBSS. An initial culturing of macrophages in the absence of FBS facilitated a selective attachment and spreading of peritoneal macrophages, while preventing lymphocytes and other cells from being attached to the culture surface. After monolayer cultures of macrophages were obtained, the cells were cultured and treated as described in the text in the presence of RPMI 1640 supplemented with 10% FBS unless otherwise indicated.
RNA isolation, cDNA synthesis, and semiquantitative analysis of cytokine mRNA levels by reverse transcriptase (RT)-PCR
Peritoneal macrophages cultured in six-well plates were primed for 6 h at 37°C with O111:B4 LPS in a range of concentrations of 0.1 to 10 ng/ml. Total RNA was extracted from the cells by TRIzol Reagent (Life Technologies, Grand Island, NY) following the manufacturer’s procedure. Equal amounts of total RNA (0.8–1.0 μg) corresponding to each priming dose were reverse transcribed using oligo(dT)16 as a primer and a complete GeneAmp RNA PCR Kit (Perkin-Elmer, Norwalk, CT). cDNA obtained after reverse transcription was amplified using specific cytokine primers and AmpliTaq DNA polymerase (Perkin-Elmer) following the protocols provided. The primers used were mouse β-actin (sense, residues 206–227, TGTGATGGTGGGAATGGGTCAG; antisense, residues 698–719, TTTGATGTCACGCACGATTTCC), mouse IL-12 p40 (sense, CAGAAGCTAACCATCTCCTGGTTTG; antisense, TCCGGAGTAATTTGGTGCTTCACAC), mouse IL-10 (sense, residues 226–249, GTGAAGACTTTCTTTCAAACAAAG; antisense, residues 476–499, CTGCTCCACTGCCTTGCTCTTATT), mouse IFN-β (sense, residues 16–36, CTCCAGCTCCAAGAAAGGACG; antisense, residues 457–477, GAAGTTTCTGGTAAGTCTTCG), and mouse IFN-γ (sense, residues 130–153, TACTGCCACGGCACAGTCATTGAA; antisense, residues 511–534, GCAGCGACTCCTTTTCCGCTTCCT). PCR amplification was performed using 30 cycles of 2 min of denaturation at 94°C, 2 min of annealing at 60°C, and 7 min of extension at 72°C. Samples were subjected to electrophoresis on 1% agarose gels, stained with ethidium bromide, and photographed.
Cytokine production by macrophages was analyzed in culture supernatants after a 24-h cell stimulation with LPS. TNF-α bioactivity in cell culture supernatants was measured by a cytotoxicity assay on L929 cells essentially as described previously (16). A specific ELISA was used for determination of IL-12, IL-6, and IL-10. A mouse IL-10 ELISA Kit obtained from Genzyme was used for detection of IL-10 (sensitivity of ELISA: 15 pg/ml). IL-6 was measured by ELISA using a specific pair of anti-mouse IL-6 mAbs and recombinant mouse IL-6 purchased from PharMingen (sensitivity of assay: 50 pg/ml). Recombinant IL-12 and anti-mouse IL-12 mAbs were provided by Genzyme and used for the determination of IL-12. Briefly, 96-well plates (Immulon 1; Dynatech Laboratories, Chantilly, VA) were coated overnight at 4°C with either 100 μl/well of capture anti-IL-6 or anti-IL-12 mAb at 1 μg/ml in 50 mM borate buffer, pH 8.5. Nonspecific binding was blocked with PBS supplemented with 0.1% Tween-20 and 10% FBS at 37°C for 1 h. Appropriately diluted samples were added in triplicates together with a serial twofold dilution of corresponding recombinant cytokine for the creation of a standard curve. After incubation at 37°C for 2 h, plates were washed with PBS/Tween-20 buffer, and 100 μl of corresponding biotinylated mAbs at a concentration of 1 μg/ml were added to each well and incubated for an additional hour at 37°C. After extensive washing with PBS/Tween-20, horseradish peroxidase-conjugated avidin (Pierce, Rockford, IL) was added to each well and incubated for 30 min at 37°C. Finally, 3,3′,5,5′ Tetramethylbenzidine substrate solution (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added and OD was measured at 405 nm after 15 min of developing at room temperature. Cytokine concentrations in test samples were calculated by comparison with a corresponding standard curve. The sensitivity of IL-12 ELISA was 20 pg/ml.
NO in 24-h conditioned culture supernatants was measured as amounts of nitrite, a stable product of NO decay, using Greiss reagent (17).
Priming with LPS modulates different phenotypes in macrophages
Previously, we have shown that priming of mouse thioglycollate-elicited peritoneal macrophages with threshold LPS concentrations results in a dose-dependent differential regulation of TNF-α, IL-6, and NO production by the cells (14, 15, 16). Thus, when freshly isolated and otherwise naïve macrophages were primed with low LPS concentrations for 6 h and then restimulated with 1 μg/ml of LPS for 24 h, a reciprocal regulation of TNF-α and NO responses in the cells was observed (Fig. 1⇓A). As a next step to understanding the molecular mechanism of this phenomenon, we have assessed the stability of modulated TNF-α and NO responses in macrophages. These experiments were conducted to test the hypothesis that the commitment of macrophages to either the TNF-α or NO phenotype is entirely dependent on the reprogramming stage so that the relative balance of TNF-α/NO is maintained constant upon subsequent stimulation with various effective concentrations. In these experiments, three separate sets of peritoneal macrophages were primed with either 100 pg/ml, 500 pg/ml, or 5 ng/ml of LPS for modulation of either a TNF-α- or NO-dominant type of response, respectively. Primed macrophages were then washed and restimulated with various effective concentrations of LPS in the range of concentrations of 20 to 2000 ng/ml. TNF-α and NO levels in the cell culture supernatants were determined after a 24-h stimulation with LPS and, thus, a pair of TNF-α and NO values corresponding to each effective LPS dose was obtained. Figure 1⇓B shows experimental curves reflecting a relative balance of TNF-α and NO produced by LPS-stimulated macrophages that were primed with the three different LPS doses. The data shown in Figure 1⇓B strongly suggest that, regardless of the secondary LPS dose used for stimulation of the cells, macrophages primed with the same LPS pretreatment concentration express a similar response in the sense that the relative balance of TNF-α and NO produced by the cells is maintained at the same level.
Role of soluble factors in LPS-induced modulation of macrophage phenotypes
In an attempt to identify the role of soluble factors/mediators in supernatants of LPS-primed macrophages that might be responsible for modulation of macrophage phenotypes, two identical sets of naïve peritoneal macrophages were obtained. One set of macrophages was maintained in standard culture conditions of RPMI 1640 supplemented with 10% FBS, while another set of the cells was primed with various LPS amounts for 6 h. Cell culture supernatants at each LPS priming dose were individually collected and filtered to remove cells and debris particles. The LPS concentrations in the samples were then adjusted by adding stock LPS to generate culture supernatants containing 1 μg/ml of LPS. Then, the supernatants were transferred to unprimed naïve macrophages for a direct LPS stimulation in the presence of secretory products produced by LPS-primed cells. The data presented in Figure 2⇓ would support the concept that LPS-dependent TNF-α and NO responses in unprimed macrophages may be modulated by soluble factors present in supernatants from LPS-primed cells.
Modulation of LPS-dependent responses in macrophages with recombinant cytokines
To further ascertain the potential role of a cytokine regulatory network in driving polarization of macrophage phenotypes, naïve macrophages were primed with various concentrations of different recombinant cytokines for 6 h before challenge with LPS. The effects of this cytokine pretreatment on subsequent TNF-α, IL-6, and NO production by LPS-stimulated cells were then analyzed. In the experiments presented in Figure 3⇓, we tested the effects of mouse rIFN-γ in the range of concentrations of 0.1 to 10 U/ml on the modulation of LPS-dependent activation of naïve macrophages. Pretreatment with IFN-γ strongly up-regulated TNF-α (Fig. 3⇓A) in a dose-dependent fashion and NO (Fig. 3⇓B) production by LPS-stimulated macrophages. Interesting, the effect of IFN-γ pretreatment on LPS-dependent IL-6 release was insignificant. A neutralizing anti-IFN-γ mAb at a concentration of 1 μg/ml partially inhibited the effects of IFN-γ pretreatment (Fig. 3⇓A), whereas the control isotype-matched IgG had little effect (data not shown). Although anti-IFN-γ mAbs and other neutralizing anti-cytokine mAbs (see below) taken at concentrations of 1 μg/ml only partially inhibited cytokine priming effects, the use of this concentration was dictated by the findings that higher Ab doses resulted in highly unacceptable background, e.g., detectable cytokine and NO levels in supernatants of cells exposed to the cytokine/Ab mixture without LPS stimulation (data not shown). Interesting, the presence of anti-cytokine Abs during stimulation of the primed macrophages with LPS did not substantially affect modulation of phenotypic responses, suggesting the pivotal role of cytokine regulatory mechanism(s) operating during the priming stage.
Priming of naïve macrophages with rIL-12 had a modest potentiating effect on both LPS-dependent TNF-α (Fig. 4⇓A) and IL-6 (Fig. 4⇓B) responses in the cells. However, IL-12 pretreatment was ineffective in modulation of the NO response (Fig. 4⇓B). The specificity of IL-12 priming effects on macrophages was confirmed by inhibition of its effects with neutralizing anti-IL-12 mAb (Fig. 4⇓A).
Finally, priming with rIL-10 dose dependently down-regulated TNF-α and IL-6 production in cytokine-primed cells and slightly up-regulated NO release upon restimulation of macrophages with LPS (Fig. 5⇓). In addition, rIL-10, added in combinations with IL-12 or IFN-γ taken at their optimal priming doses, was still capable of inhibiting in a dose-dependent manner the priming effects of these cytokines on TNF-α and NO production by macrophages (data not shown).
Differential regulation of IL-10 and IL-12 production by LPS-primed macrophages
Due to the common permissive/inhibitory relationship of cytokines in the control of cellular production of each other, we next evaluated the levels of IL-10, IL-12, and IFN-γ in 24-h supernatants of LPS-primed and LPS-stimulated macrophages to assess the potential role of these cytokines in modulation of TNF-α and NO responses in LPS-primed macrophages. Although a sensitive ELISA was performed to measure the amounts of IFN-γ (detection limit 25 pg/ml) in culture supernatants, no detectable levels of IFN-γ were found in the test samples (data not shown). In contrast, priming with LPS differentially and dose dependently regulated production of IL-10 and IL-12, which were reciprocally regulated in LPS-primed macrophages (Fig. 6⇓). In addition, data shown in Figure 6⇓ indicate that the peak of IL-12 production approximately coincides with the peak of TNF-α production by LPS-primed macrophages, whereas the profile of IL-10 release parallels the one for NO response (Fig. 1⇑A).
Effects of neutralizing anti-cytokine mAbs on LPS-induced priming of macrophages
To further characterize the cytokine regulatory pathway(s) involved in LPS-dependent priming of macrophages, we tested the capacity of neutralizing anti-IL-10, anti-IL-12, and anti-IFN-γ mAbs to influence the LPS-induced priming process. In this set of experiments, macrophages were primed with LPS in the presence of 20 μg/ml of each neutralizing mAb added alone. Then, after extensive washing, cells were restimulated with an effective LPS dose and the amounts of TNF-α and NO were determined in culture supernatants 24 h later. The data presented in Figure 7⇓A show that neutralization of IL-12 and IFN-γ during the LPS priming stage attenuated TNF-α production by LPS-stimulated macrophages. In contrast, neutralization of IL-10 resulted in TNF-α release at LPS priming concentrations that otherwise strongly inhibited TNF-α production by the cells. When the effects of neutralizing mAbs on NO response of LPS-primed macrophages were analyzed, we found that anti-IFN-γ and anti-IL-10 mAb inhibited NO production by LPS-primed macrophages, whereas neutralization of IL-12 boosted LPS-dependent NO responses in the cells (Fig. 7⇓B). These data would indicate the potential role of IL-10, IL-12, and IFN-γ in modulation of both TNF-α and NO phenotypes in LPS-primed macrophages.
RT-PCR analysis of cytokine mRNA expression during the LPS priming stage
That macrophages were exposed to the tested anti-cytokine-neutralizing mAbs only during the LPS priming stage would suggest that polarization of the development of macrophage phenotypes may occur during the priming stage and be mediated by an LPS-activated cytokine network. To test this experimental hypothesis, we analyzed cytokine levels in culture supernatants of macrophages that were only primed with threshold LPS concentrations for 6 h. However, we were unable to detect ELISA-measurable amounts of IL-10, IL-12, TNF-α, and IFN-γ in collected supernatants. We therefore analyzed the differential expression of cytokine mRNA using RT-PCR. In these experiments, naïve macrophages were primed with various concentrations of LPS for 6 h before total cellular RNA was isolated, reverse transcribed, and specifically amplified using primers for different mouse cytokines. Data presented in Figure 8⇓ show a differential dose-dependent regulation of cytokine mRNA expression in LPS-primed macrophages. Expression of IL-12 mRNA was found to be up-regulated at LPS priming concentrations that overlapped with the LPS priming doses we previously established to modulate an increased TNF-α production in macrophages (TNF-α phenotype). In contrast, IL-10 mRNA, IFN-β mRNA, and IFN-γ mRNA were detectable only at high LPS priming doses of 1 to 10 ng/ml and overlapped with LPS priming concentrations previously established to preferentially induce the NO phenotype in macrophages. Interestingly, analysis of expression of the TNF-α message revealed comparable levels of TNF-α mRNA in macrophages primed with different LPS reprogramming doses in the range of 0.1 to 10 ng/ml, suggesting a posttranscriptional mechanism governing TNF-α production by the cells.
In the present study, we provide experimental evidence for the hypothesis that modulation of specific responses in LPS-primed macrophages is dictated by a cytokine regulatory mechanism(s) derived through a reciprocal regulation of IL-10 and IL-12 production in the cells. We found that an efficient LPS-dependent modulation of TNF-α and NO phenotypes in macrophages may well depend on autocrine/paracrine signals mediated via the release of specific soluble factors/mediators by the cells during the LPS priming stage. To confirm the potential role of cytokines in the modulation of LPS priming effects, we evaluated LPS-dependent TNF-α and NO responses in cytokine-primed macrophages pretreated with different recombinant cytokines before stimulation with LPS. Our data strongly suggest that an exposure of naïve macrophages to individual cytokines such as IFN-γ, IL-12, and IL-10 or a combination of these cytokines may differentially modulate cell responsiveness to LPS. Therefore, among other regulatory pathways, a cytokine network may well represent a potential mechanism(s) controlling the bias of specific macrophage phenotypes induced by LPS priming. Analysis of cell-derived IL-10 and IL-12 concentrations in culture supernatants of LPS-primed and LPS-stimulated macrophages revealed a significant correlation between the levels of IL-12 and TNF-α as well as between the levels of IL-10 and NO. This finding establishes a necessity between modulation of proinflammatory TNF-α phenotype of response and up-regulated production of IL-12 in LPS-primed macrophages, whereas modulation of the NO phenotype may well be associated with up-regulated production of IL-10 and its antiinflammatory effects. Furthermore, neutralization of endogenously produced IL-10 and IL-12 with anti-IL-10 and anti-IL-12 mAbs significantly affected LPS-dependent responses in LPS-primed cells, further suggesting the pivotal role of IL-12 and IL-10 in the modulation of TNF-α and NO phenotypes in macrophages. In addition, we found differential and dose-dependent regulation of mRNA expression for IL-12 and IL-10 in LPS-primed cells. Therefore, it is reasonable to conclude that endogenously produced IL-12 may promote and control a proinflammatory TNF-α phenotype in LPS-primed macrophages by up-regulating TNF-α production and inhibiting NO release. In contrast, cell-derived IL-10 may well control modulation of an NO phenotype associated with antiinflammatory cell responses by up-regulating NO release and limiting TNF-α production in LPS-primed macrophages. In addition, up-regulated expression of IFN-β and IFN-γ mRNAs in LPS-primed macrophages and recently reported synergistic effects of these cytokines on LPS-dependent activation of inducible NO synthase suggest the potential role of IFNs in the modulation of the NO phenotype in LPS-primed macrophages.
Although IL-10 inhibits in vitro activation and NO production by macrophages, our results suggest a potential regulatory role of IL-10 in the modulation of the NO phenotype in LPS-primed macrophages. It has yet to be established whether IL-10 directly modulates the NO phenotype in LPS-primed macrophages or whether this correlation is circumstantial and other factors/cytokines play a key role in the complex regulatory process controlling macrophage reprogramming. Additional experiments on macrophages isolated from IL-10, IL-12, and IFN-γ knockout mice will allow the dissection of the specific role of each of these cytokines in the modulation of different phenotypes in LPS-primed macrophages. However, experimental data available support our hypothesis that the commitment of macrophages to either the TNF-α or NO phenotype of the LPS-dependent response occurs during the priming stage and is mediated by a cytokine regulatory pathway(s) derived through a reciprocal induction of IL-12 and IL-10.
Due to the crucial role of IL-10 and IL-12 in the polarization of T cell responses, a reciprocal control of IL-12 and IL-10 production in LPS-primed macrophages such as APCs may well be instrumental for developing host immune responses in general. The adaptive response to infections is characterized by the differentiation of Th cells into either the Th1 or Th2 phenotype, which then favors cell-mediated immunity and humoral responses, respectively (18, 19, 20, 21). Th1 development is positively regulated by two major cytokines, IL-12 and IFN-γ, and, in general, results in localization and cure of the infection (22, 23, 24, 25, 26). In contrast, IL-10, an antiinflammatory cytokine produced by activated macrophages/monocytes, synergizes with IL-4 in the induction of Th2-type lymphocyte development and is found to be associated with chronic, progressive infections (27, 28).
Macrophages can produce cytokines that are vital for T cell development, such as IL-10 and IL-12, as well as some amounts of IFN-γ, as suggested in earlier studies (29). Evidently, if a reciprocal production of IL-12 and IL-10 in macrophages is achieved, it would be expected to provide a cytokine microenvironment facilitating development of either the Th1 or Th2 subset, respectively. We therefore hypothesize that the plasticity of APC-derived cytokine production as shown in the data presented here for macrophages, and controlled by dose-dependent LPS priming of the cells, may dictate the outcome of APC-T cell interactions. We speculate that, depending on the dominant type of cytokine response, e.g., IL-12 or IL-10, acquired by macrophages after priming with LPS, primed macrophages functioning as APC would provide an accessory signal for the development of either the Th1 or Th2 subset, respectively. Specifically, up-regulated production of IL-12 can directly (30, 31, 32) or, most likely, via synergism with TNF-α-dependent induction of IFN-γ (33, 34), control the development of Th1 type. In contrast, macrophage phenotype characterized by overproduction of IL-10 may well favor proliferation of the Th2 type of T cells through a synergistic action with IL-4 (27, 28) and antagonistic effects on Th1-type cytokine production (35, 36). Furthermore, the differential IL-10 and IL-12 production in LPS-primed macrophages such as APCs is primarily dependent on the load of Ag/LPS, since LPS priming in macrophages is a dose-dependent process. Priming of macrophages is induced by threshold LPS concentrations and, therefore, may take place during the initial stage of disease at the “gate of infection.” Importantly, newly recruited phagocytes/APCs could be “educated” by already primed macrophages through the specific cytokine milieu produced by the earlier arrived APCs. Further investigation of the mechanism(s) controlling reciprocal production of IL-10 and IL-12 in LPS-primed macrophages and its potential role in modulation of differentiation of T cell precursors into functionally distinct Th1 and Th2 subsets of T lymphocytes is under progress in our laboratory.
We thank Eleanor Zuvanich for expert technical assistance in preparation of L929 cells for TNF bioassay.
↵1 This work was supported by National Institute of Health Grants POI CA54474 and R37AI23447. The support of the Kansas Masonic Foundation is also gratefully acknowledged. The work was done in partial fulfillment of the requirements for the “Mentorship Program” for R.B. in the Blue Valley School District, Overland Park, KS and for the Ph.D. degree for A.A. in United Laboratories, Inc., Manila, Philippines.
↵2 Current address: Dr. Arlene Alipio, United Laboratories, Inc., PO Box 3594, Manila, Philippines.
↵3 Address correspondence and reprint requests to Dr. Alexander Shnyra, The University of Kansas Medical Center, Department of Microbiology, Molecular Genetics and Immunology, Kansas City, KS 66160.
4 Abbreviations used in this paper: NO, nitric oxide; RT, reverse transcriptase.
- Received July 17, 1997.
- Accepted December 16, 1997.
- Copyright © 1998 by The American Association of Immunologists