We report that the addition of human macrophage inflammatory protein-3β (MIP-3β) to cultures of human PBMCs that have been activated with LPS or PHA results in a significant enhancement of IL-10 production. This effect was concentration-dependent, with optimal MIP-3β concentrations inducing more than a 5-fold induction of IL-10 from LPS-stimulated PBMCs and a 2- to 3-fold induction of IL-10 from PHA-stimulated PBMCs. In contrast, no significant effect on IL-10 production was observed when 6Ckine, the other reported ligand for human CCR7, or other CC chemokines such as monocyte chemoattractant protein-1, RANTES, MIP-1α, and MIP-1β were added to LPS- or PHA-stimulated PBMCs. Similar results were observed using activated purified human peripheral blood monocytes or T cells. Addition of MIP-3β to nonactivated PBMCs had no effect on cytokine production. Enhancement of IL-10 production by MIP-3β correlated with the inhibition of IL-12 p40 and TNF-α production by monocytes and with the impairment of IFN-γ production by T cells, which was reversed by addition of anti-IL-10 Abs to the cultures. The ability of MIP-3β to augment IL-10 production correlated with CCR7 mRNA expression and stimulation of intracellular calcium mobilization in both monocytes and T cells. These data indicate that MIP-3β acts directly on human monocytes and T cells and suggest that this chemokine is unique among ligands binding to CC receptors due to its ability to modulate inflammatory activity via the enhanced production of the anti-inflammatory cytokine IL-10.
The members of the chemokine superfamily are integrally involved in a variety of immunological and inflammatory functions via their ability to induce leukocyte trafficking, regulate hematopoiesis, regulate angiogenesis, and suppress the infection of HIV (1, 2). At least two subfamilies of chemokines have been identified, the CC and CXC chemokines, based on the positioning of two amino-terminal cysteine residues (1, 2). All chemokines exert their activity by binding to 7-transmembrane G protein-coupled receptors, which are expressed on a variety of leukocyte populations. The CC chemokine macrophage inflammatory protein-3β (MIP-3β)2 (CKβ-11/EBV-induced molecule-1) is produced in lymphoid tissues and at sites of inflammation, binds exclusively to CCR7 on activated T and B cells and mature dendritic cells, and induces the chemotaxis of these cells (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). MIP-3β was also found to selectively induce the rapid adhesion of lymphocytes to purified ICAM-1 under flow conditions at 1 μM (12).
Although there have been a number of reports suggesting that cytokines such as IL-10 and IFN-γ can modulate both chemokine production and chemokine receptor expression by leukocytes (13, 14, 15, 16, 17, 18, 19), there have been a limited number of investigations of the effect of chemokines on leukocyte cytokine production. At an in vitro concentration of 1 μM, the CC chemokine RANTES by itself was shown to induce IL-2 and IL-5 production, augment proliferation, and enhance the expression of a variety of activation and adhesion molecules on a human T cell clone and on primary T cells (20, 21). Addition of MIP-1α, MIP-1β, RANTES, or monocyte chemoattractant protein-1 (MCP-1) was shown to costimulate human primary T cells and T cell clones in an IL-2-dependent manner (22). In addition, MIP-1α and MCP-1 were able to differentially augment IFN-γ and IL-4 production by murine OVA-stimulated TCR-transgenic T cells (23). Such studies are important, because identifying the role of chemokines in modulating cytokine synthesis within the inflammatory microenvironment may be useful for understanding the pathogenesis of acute and chronic inflammatory diseases.
Here we report that MIP-3β is able to enhance IL-10 production by LPS- and PHA-stimulated PBMCs. Increased IL-10 production in these cultures corresponded with reduced levels of proinflammatory cytokines. This effect is also seen with purified monocytes and T cells and correlates with induction of intracellular calcium flux in these cell populations. These results indicate that MIP-3β may be capable of altering the consequence of inflammatory responses via the induced production of IL-10, a cytokine with well-documented anti-inflammatory activities (13).
Materials and Methods
Cell preparation, stimulation, and cytokine measurements
Human PBMCs were prepared by Ficoll-Hypaque centrifugation and stimulated with 2 μg/ml PHA (Murex Diagnostics, Dartford, U.K.) or 100 ng/ml LPS (Sigma, St. Louis, MO) in Yssel’s medium (Gemini Bioproducts, Calabasas, CA) as described previously (24). All chemokines were purchased from Peprotech (Rocky Hill, NJ) or R&D Systems (Minneapolis, MN) and added at the initiation of culture. After 40 h, culture supernatant was collected and cytokine levels were determined by ELISA (R&D Systems). Cell viability, as measured by trypan blue dye exclusion, was unaffected by any of the treatment conditions. Purified peripheral blood monocytes were prepared by centrifugation through a discontinuous Percoll gradient as described previously (25) and cultured with LPS. This protocol resulted in >80% CD14+ monocytes by flow cytometry, with the remaining cells being CD3−, CD4−, CD8−, CD19−, CD20−, and CD56−. In some experiments, to detect chemokine receptor expression by RT-PCR, monocytes were purified using NycoPrep gradients, as per the manufacturer’s directions (Accurate, Westbury, NY). This procedure reproducibly resulted in >90% monocytes. Human peripheral blood T cells were obtained by negative selection on Cellect Plus columns (Biotex, Edmonton, Alberta, Canada) supplemented with additional anti-CD16 and anti-CD56 Abs to ensure depletion of NK cells. Final cell populations were 90–95% CD4+ or CD8+ T cells and were cultured in 96-well plates previously coated with 5 μg/ml anti-CD3 (clone UCHT-1, PharMingen, San Diego, CA). Anti-CD28 Ab was added at 1 μg/ml. In some experiments, anti-IL-10 (clone 19F1) or isotype-matched rat IgG2a was added to the cultures at 2 μg/ml. In some figures, cytokine data are presented as percent of control to normalize our panel of donors; treatment groups that received only LPS, PHA, or anti-CD3/anti-CD28 are represented as 100%. The absolute levels of cytokines in these groups are described in each figure legend. All flow cytometric analyses were performed using a FACScalibur and CellQuest analysis software (Becton Dickinson Immunocytometry Systems, Mountain View, CA).
RT-PCR for chemokine receptors
Total cellular RNA was prepared from human PBMCs, T cells, or monocytes, reverse-transcribed as described previously (26), and subjected to PCR for 30 cycles at 94°C for 0.5 min, 57.5°C for 1 min, and 72°C for 1 min. For CCR7, a 573-bp product was obtained using the following primers: forward, 5′-CGCGTCCTTCTCATCAGCAA-3′; reverse, 5′-GTGCCGACAGGAAGACCACT-3′. For the housekeeping gene GAPDH, a 452-bp product was obtained using the following primers: forward, 5-AACACAGTCCATGCCATCAC-3′; reverse, 5′-TCCACCACCCTGTTGCTGTA-3′. Preliminary experiments were performed to ensure the linearity of the PCRs. For photographic reproduction, photographs were optically scanned as described previously (24).
Measurement of intracellular calcium flux
Monocytes that had been purified by the NycoPrep method described above were plated in 96-well plates (Packard, Meriden, CT) in Yssel’s medium at 1.2 × 105 cells/well in the presence or absence of 100 ng/ml LPS and allowed to adhere overnight. Nonadherent cells were removed, and the adherent cells were washed three times with PBS, loaded with 4 μM of Fluo-3 (Molecular Probes, Eugene, OR), washed with fluorometric imaging plate reader (FLIPR) buffer, and analyzed for intracellular calcium mobilization using the FLIPR system (Molecular Devices, Palo Alto, CA) as described previously (27).
To assess the effect of CC chemokines on human peripheral blood cell cytokine production, PBMCs were prepared and incubated with PHA or LPS in the presence or absence of various concentrations of MIP-3β, 6Ckine, MIP-1α, MIP-1β, RANTES, or MCP-1. As shown in Fig. 1,A, addition of MIP-3β to cultures of human PBMCs stimulated with LPS resulted in a concentration-dependent increase in IL-10 production, with a statistically significant increase in IL-10 levels of >2-fold (∼250% of control) observed at 1000 ng/ml (∼125 nM) and a >5-fold enhancement of IL-10 levels observed at 8000 ng/ml (1 μM) of MIP-3β. In contrast, the addition of equivalent concentrations of 6Ckine had only a slight effect on IL-10 production, and the addition of MIP-1α, MIP-1β, RANTES, or MCP-1 did not affect IL-10 production from these LPS-stimulated cultures (Fig. 1,A). Addition of MIP-3β to human PBMC cultures stimulated with the T cell mitogen PHA also enhanced IL-10 production in a concentration-dependent fashion, whereas none of the other chemokines tested, including 6Ckine, had any activity (Fig. 1 B). As seen in the LPS-stimulated cultures, MIP-3β caused a statistically significant, concentration-dependent increase in IL-10 production in PHA-activated PBMC cultures, with a 2- to 3-fold enhancement observed at a MIP-3β concentration of 8000 ng/ml. No change in the expression of activation markers such as CD69 or CD25 was observed in PHA-stimulated PBMC cultures to which MIP-3β was added, and only a modest (10–15%) reduction in proliferation in a subset of donors was observed (data not shown). These data indicate that MIP-3β may be selective among chemokines that bind to CC receptors due to its ability to augment IL-10 production from stimulated PBMCs. Addition of MIP-3β to unstimulated cultures had no effect on constitutive IL-10 production (data not shown).
IL-10 has been demonstrated previously to suppress a number of proinflammatory activities, including cytokine production, under a variety of circumstances (13). To directly examine whether the increase in IL-10 production induced by culture with MIP-3β led to an inhibition of proinflammatory cytokines, we measured the levels of IL-12 p40 and TNF-α in these cultures. As indicated in Fig. 2,A, IL-12 p40 and TNF-α levels were inhibited in a concentration-dependent manner in LPS-stimulated PBMC cultures, with maximal inhibition of ∼50% at 1000 ng/ml MIP-3β. Similarly, MIP-3β caused a 25–40% reduction in IFN-γ levels in PHA-stimulated PBMC cultures (Fig. 2,A). Treatment with 8000 ng/ml MIP-3β did not lead to additional inhibition of the levels of these cytokines. Addition of anti-IL-10 to LPS- or PHA-stimulated cultures was able to reverse the inhibition of IL-12, TNF-α, and IFN-γ synthesis (Fig. 2,B), confirming that the MIP-3β-induced inhibition of these proinflammatory cytokines is mediated via IL-10. In the anti-IL-10-treated cultures, we observed an enhancement of the level of these cytokines relative to untreated cultures (Fig. 2 B, labeled as no MIP-3β), probably due to neutralization of endogenously produced IL-10, which serves to regulate the synthesis of these mediators.
To extend these observations, we evaluated the ability of MIP-3β to modulate IL-10 production on purified populations of monocytes or T cells. Similar to what we observed with bulk PBMC cultures, MIP-3β, but not 6Ckine, was able to induce a significant increase in IL-10 production from LPS-stimulated monocytes as well as anti-CD3 plus anti-CD28-stimulated purified T cells (Fig. 3). Again, this activity was correlated with a significant inhibition of IL-12 and TNF-α production by monocytes and of IFN-γ by T cells (data not shown).
The above data suggest that MIP-3β mediates its IL-10-enhancing activity directly on monocytes and T cells. The observation that MIP-3β acts on monocytes was surprising, because MIP-3β was unable to induce monocyte chemotaxis (5, 9), suggesting that CCR7, the only reported receptor for MIP-3β, is not expressed by human monocytes. To directly the evaluate the role of CCR7 on monocytes, we evaluated its expression by RT-PCR and the induction of intracellular calcium flux by MIP-3β and 6Ckine. As illustrated in Fig. 4, we detected CCR7 mRNA in both freshly isolated and LPS- and PHA-treated PBMCs, as well as in fresh and LPS-treated monocytes and freshly prepared and anti-CD3 plus anti-CD28-activated T cells by RT-PCR. Addition of MIP-3β to purified monocytes that were incubated with LPS and allowed to adhere overnight resulted in the stimulation of intracellular calcium mobilization in a concentration-dependent fashion (Fig. 5,A). 6Ckine was also able to induce calcium flux in these adherent cells as well (Fig. 5 B). These data suggest that functional CCR7 is expressed on LPS-stimulated human monocytes and that the MIP-3β-induced enhancement of IL-10 synthesis may be mediated via this receptor.
The data presented here suggest that MIP-3β is selective among chemokines binding to CC receptors because of its ability to augment IL-10 production from activated human monocytes and T cells. This effect is concentration-dependent and results in a statistically significant enhancement of IL-10 biosynthesis. This MIP-3β-induced IL-10 production results in an inhibition of the production of proinflammatory cytokines such as IL-12 p40, TNF-α, and IFN-γ, and this inhibition is reversed by coincubation with anti-IL-10. In contrast, 6Ckine, the other known ligand for human CCR7, was unable to significantly modulate cytokine production. The effects of MIP-3β that we observed with unfractionated PBMCs were seen in purified monocyte and T cell cultures as well, and correlated with CCR7 mRNA expression and MIP-3β-induced intracellular calcium flux, suggesting that this chemokine is able to signal directly on these cell populations. Although CCR7, the only documented receptor for MIP-3β, has previously been reported to be present on T cells, it has not previously been demonstrated on human monocytes (3, 5, 9, 10, 28). Here we report for the first time that functional CCR7 is expressed on human monocytes that have been treated with LPS overnight. Overall, our data imply that the MIP-3β-induced enhancement of IL-10 production in human monocytes and T cells may be mediated via signaling through CCR7.
Our observations that MIP-3β can enhance the production of IL-10, a potent immunosuppressive cytokine (13), were somewhat surprising, given that chemokines are generally thought to play an important role in promulgating, rather than suppressing, inflammation. The present results suggest that under some circumstances, MIP-3β may act to limit rather than amplify inflammatory sequelae, perhaps at the later stages of an immune response. Given that 6Ckine, the other known ligand for human CCR7 (29, 30, 31, 32, 33), MIP-1α, MIP-1β, RANTES, and MCP-1 were not able to modulate IL-10 production, MIP-3β may play a unique role during the course of an inflammatory response via its ability to induce IL-10. Interestingly, Rossi et al. (4) previously reported that anti-IL-10 treatment of activated human monocytes significantly up-regulates MIP-3β mRNA expression. These data, combined with our own, suggest a regulatory mechanism by which MIP-3β enhances IL-10 biosynthesis, which then serves to suppress the production of chemokines and proinflammatory cytokines (13). Such a mechanism may be regulated, in part, by a primary signaling event (e.g., LPS- or TCR-mediated) to prevent inappropriate IL-10 production. A role for MIP-3β-induced IL-10 production in regulating events in secondary lymphoid tissues and/or modulating B cell activation (9, 10) must be considered as well.
We found statistically significant increases in IL-10 production at 200-1000 ng/ml of MIP-3β (25–125 nM), whereas maximal IL-10 levels were observed at a MIP-3β concentration of 8000 ng/ml (∼1 μM). Preliminary experiments have revealed similar increases in steady-state IL-10 mRNA levels as well. These data are consistent with previous reports indicating that 1 μM of MIP-3β is able to induce lymphocyte adhesion to ICAM-1 under flow conditions (12), and that RANTES at 1 μM is able to enhance T cell proliferation, cytokine production, and IL-2R and adhesion molecule expression (20, 21). Chemokine concentrations in the micromolar range within inflammatory microenvironments via sequestration by cell surface glycosaminoglycans and local presentation to cells have been postulated previously (20), based on studies indicating that chemokines such as IL-8, growth-related oncogene (GRO), platelet factor 4, RANTES, IFN-γ-inducible protein-10, and MCP-1 bind well to proteoglycans (34, 35, 36, 37, 38, 39, 40, 41, 42, 43). It is unclear whether MIP-3β binds to glycosaminoglycans in a similar fashion. Alternatively, MIP-3β may aggregate under the conditions used here, as has been suggested for RANTES (20, 44), rather than remain as an unaggregated monomer or dimer (45, 46), resulting in a significant reduction in its effective concentration.
The finding that MIP-3β was active on purified human monocytes was surprising and led us to examine the expression of CCR7 on these cells. By RT-PCR, we were able to detect CCR7 mRNA in unstimulated and activated PBMCs, monocytes, and T cells (Fig. 4). This finding differs from previous data indicating that CCR7 mRNA is not expressed in human monocytes, but is restricted to activated T cells, B cells, and mature dendritic cells (3, 5, 6, 7, 8, 9, 10, 11). We went on to investigate MIP-3β-induced signaling in these purified monocytes by evaluating chemokine-induced intracellular calcium mobilization. Both MIP-3β (Fig. 5,A) and 6Ckine (Fig. 5,B) induced a profound increase in intracellular calcium flux in adherent monocytes. The response induced by MIP-3β and 6Ckine was seen on adherent monocytes in the presence (Fig. 5) or absence (our unpublished observations) of LPS stimulation. In contrast, we observed only occasional weak calcium mobilization induced by MIP-3β or 6Ckine when freshly isolated monocytes were examined (data not shown), consistent with prior observations that these chemokines do not mediate monocyte chemotaxis (5, 9) or induce it very weakly (47). In comparison, MCP-1 induced a strong calcium flux on fresh monocytes but was unable to stimulate a response on LPS-stimulated cells (data not shown). These data suggest that overnight adherence of monocytes provides a requisite signal(s) leading to functional CCR7 expression. Analogous observations have been made with dendritic cells, which require specific signals such as anti-CD40 ligation or LPS activation to express functional CCR7 (6, 7, 8, 9, 10, 11). Interestingly, MIP-3β has been shown previously to serve as a chemoattractant (and presumably induces intracellular calcium flux) for human bone marrow macrophage progenitor cells (48).
Although our data indicate that MIP-3β-induced IL-10 production by monocytes and T cells correlates with functional CCR7 expression, we have not been able to directly address the role of CCR7 in this response, in part due to the lack of availability of a neutralizing anti-human CCR7 Ab. Such an Ab may be useful as well for phenotypic analysis of cell populations, as has been reported recently for dendritic cells (49) and T cells (50). In addition, it is unclear why MIP-3β, but not 6Ckine, demonstrated this IL-10-enhancing activity, even though both of these chemokines bind with high affinity to CCR7 and induce an equivalent intracellular calcium flux. To our knowledge, this may be the first demonstration of a differential effect mediated through this receptor, although prior experiments have primarily focused on its role in chemotaxis. Differences in signaling pathways induced by these ligands, perhaps due to distinct ligand binding sites, may account for these findings. Presently, any relationship between ligand-induced intracellular calcium mobilization and IL-10 production in these cells remains unclear. It remains formally possible that the effect of MIP-3β on IL-10 production may be mediated by a receptor distinct from CCR7. Such a receptor is likely to be novel, because the only reported receptor for MIP-3β is CCR7 (1, 2, 3).
Our observations suggest that of all the chemokine ligands binding to CC receptors, MIP-3β plays a central role in mediating inflammatory processes by its ability to both chemoattract leukocytes and restrict an ongoing response by enhancing the production of IL-10. Thus, these data extend the list of chemokines capable of modulating immune responses by activities other than inducing chemotaxis (20, 21, 22, 23). Future experiments will examine the signaling pathways (1, 20, 51) mediating the MIP-3β-induced modulation of IL-10 biosynthesis.
We thank Drs. Loretta Bober and Paul Zavodny for helpful discussions and for reviewing the manuscript.
Abbreviations used in this paper: MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein.