LPS stimulated B-1 cell polyclonal in vivo IgM responses depend on IL-4 release by invariant Vα14+Jα18+ NKT (iNKT) cells. The IgM Abs can recruit effector T cells to mediate contact sensitivity. LPS activates the B-1 cell response just 1 day later, and depends on CD1d, iNKT cells, IL-4, TLR4, and MyD88. LPS in vivo and in vitro stimulates rapid preferential production of IL-4 in hepatic iNKT cells within 2 h. TLR4 were demonstrated in iNKT cells by flow cytometry and functional studies. Thus, innate microbial stimulation via TLR can activate iNKT cell and B-1 cell collaboration. The result is polyclonal IgM Ab responses capable of recruiting Ag-specific T cells into tissues. This may be involved in the promotion of autoimmunity by infectious agents.
Innate immunity greatly influences acquired immunity (1). Innate activation proceeds largely via recognition of conserved microbial pathogen-associated molecular patterns (PAMPs),3 such as bacterial LPS (2, 3). The microbial molecules stimulate invariant germline-encoded pattern recognition receptors, such as TLR on various host cells (3). Recognition of LPS by TLR4 is a classical example (4).
Much is now known about the adjuvant effects of microbial TLR ligands acting on APCs like dendritic cells (DC) or macrophages, to induce T cell immunity (1). We are beginning to identify important innate immune system influences on subsequently elicited effector T cell responses in vivo. Contact sensitivity (CS) and delayed-type hypersensitivity (DTH) are classical models of T cell-acquired responses that require innate immune components for elicitation. Thus, CS and DTH require complement (5, 6, 7, 8), mast cells (9, 10, 11), and platelets (12, 13) for optimal recruitment of the effector T cells into the tissues during an early 2 h “initiating” window following local Ag challenge of previously sensitized mice (14, 15, 16).
Recently, we showed that innate-like B-1 B cells also participate in this innate initiating pathway needed to recruit effector T cells (8, 17). B-1 cells are activated within just 1 h of immunization to rapidly produce IgM Abs that are present in the serum by 1 day (Refs.8 , 17 , and 48). Upon local Ag challenge, IgM Abs at the local site form complexes with the Ag to activate complement to generate C5a (5, 6, 7, 8). Elaborated C5a acts on C5a receptors to stimulate mast cell and platelet release of vasoactive TNF-α and serotonin to recruit the effector T cells locally (7, 8, 9, 10, 11, 12, 13, 18, 19, 20, 21). Furthermore, we showed recently that innate-like invariant Vα14+Jα18+ NKT (iNKT) cells were required to coactive the B-1 cells, along with specific Ag, by production of IL-4 within just minutes following immunization (Ref.22 , and R. A. Campos, M. Szczepanik, A. Itakura, M. C. Leite-de-Moraes, M. Kronenberg, and P. W. Askenase, submitted for publication).
We considered that innate immunity stimulated by PAMPs also could be involved in the elicitation of acquired T cell immunity. We hypothesized that microbial TLR ligands could stimulate polyclonal B-1 cell IgM Ab responses. Then, under certain circumstances, these Abs could act like the IgM Abs produced by B-1 cells following specific Ag immunization, to lead to recruitment of effector T cells.
In this study, we show that the TLR4 ligand LPS stimulates polyclonal B-1 cell IgM Ab responses that can lead to local recruitment of Ag-specific effector T cells to mediate the classical late T cell component of CS. A new finding is that LPS activation of the B-1 cells also depends on iNKT cell production of IL-4. We show that iNKT cells express TLR4 required for production of IL-4 to stimulate B-1 cells. Activation by LPS results in expression of IL-4 in the iNKT cells within 90 min. This very rapid LPS stimulation of iNKT cells via TLR4 is MyD88-dependent and preferentially affects hepatic iNKT cells.
We postulate that this collaboration between TLR4-expressing iNKT cells and B cells could be involved in the association of microbial infections with autoimmunity. Microbial pathogens, in addition to breaking self tolerance to generate autoreactive effector T cells, may also stimulate polyclonal Ab responses, including Abs to self components that may lead to recruitment of the autoantigen-reactive T cells into the tissues.
Materials and Methods
Specific pathogen-free male CBA/J, CBA/N-xid, TLR4 dominant-negative (C.C3Tlr LPSd), CD1d−/−), (BALB/c background); C3H/HeJ; C3H/HeN; C57BL/6; and C57BL/10ScNJ mice were obtained from The Jackson Laboratory or the National Cancer Institute (NCI; Frederick, MD). Breeders of IL-12−/− mice were provided by the National Institutes of Health (Bethesda, MD); breeders of Jα18−/− mice were provided by M. Taniguchi, Chiba University (Chiba, Japan). Mice were maintained under specific pathogen-free conditions, rested for at least 1wk before use, and used at 7–12 wk of age. Experiments were conducted according to guidelines of the Animal Care Committee of Yale School of Medicine.
The contact sensitizer trinitrophenyl chloride (TNP-Cl; picryl chloride (PCl); Nacalai Tesque) was recrystallized twice and stored protected from light. Oxazolone (OX), croton oil, Escherichia coli LPS (Sigma L2630) and PMA were obtained from Sigma-Aldrich. Anti TCR-β and anti-TCR-δ (BD Pharmingen), rabbit complement (PelFreeze), TNP-BSA (Bioscience Technologies), anti-TLR4/MD2 (MTS510; e-Biosciences), anti-IL-4 (11B11; NCI), isotype controls (BD Pharmingen), lipid A of E. coli F583 (Sigma-Aldrich), peptidoglycan of Staphylococcus aureus (Fluka), zymosan A (Sigma-Aldrich), CpG (Yale Oligonucleotide Facility), and dsRNA (Amersham Biosciences) were obtained from the manufacturer.
Immunization and elicitation of CS
Mice were contact sensitized with 150 μl of 5% TNP-Cl in absolute ethanol and acetone (4:1) on the shaved chest, abdomen, and rear footpads. CS responses were elicited on day 1 by painting ears with 10 μl of 0.4% PCl in acetone and olive oil (1:1). Ear thickness was measured with a micrometer (Mitutoyo) before challenge and then at 2 and 24 h by an observer unaware of experimental groups. Increases in ear thickness are expressed as the mean ± SE × 10−2 mm.
Liver cell preparation to obtain enriched mononuclear cells
After sacrifice, liver was PBS perfused via the portal vein until opaque, then strained (70 μm; BD Biosciences), resuspended in 100% isotonic Percoll (Amersham Pharmacia) to obtain a final concentration of 40%, and overlaid onto 60% isotonic Percoll. After centrifugation for 20 min at 900 × g at 25°C, the liver mononuclear cells (LMNC) were isolated at the interface and the 40% Percoll, and washed with RPMI 1640 (Invitrogen Life Technologies) + 5% FBS (Gemini-Bio-Products). Viability was >90%, and ∼2 × 106 LMNC were obtained per mouse.
Allophycocyanin-labeled tetrameric mouse CD1d-β2 microglobulin-α-GalCer complexes, and control “unloaded” mouse allophycocyanin-CD1d-β2 microglobulin complexes without added α-GalCer (hereafter termed mock tetramers) were used (R. A. Campos, M. Szczepanik, A. Itakura, M. C. Leite-de-Moraes, M. Kronenberg, and P. W. Askenase, submitted for publication). To evaluate tetramer binding, LMNC were resuspended in PBS-staining buffer containing 2% BSA and 0.02% NaN3, and incubated for 15 min at 4°C with blocking 2.4G2 anti-Fcγ mAb (BD Pharmingen). Cells were then stained with tetramers or mock tetramers at 25°C × 20 min and further incubated with both anti-CD4 PerCP-Cy-5.5 (clone RM4-5) and anti-TCRβ-FITC (clone H57-597) mAb (BD Pharmingen). After washing, cells were stained subsequently for intracellular cytokines, following fixation with paraformaldehyde, permeabilized with saponin and incubated with anti-IL-4-PE (clone 11B11), or anti-IFN-γ-PE (clone XMG1.2) or isotype controls (BD Pharmingen). Dead cells were excluded on the basis of forward and side scatter.
Characterization of tetramer-positive iNKT cells among the TCRβ+ and CD4+ or CD4− cells and intracellular staining were conducted using a FACSCalibur (BD Biosciences) and a minimum of 5 × 105 events were acquired. Results were analyzed using Mac CellQuest software.
In vitro activation of naive B-1 cells mixed with LMNC from mice treated in vivo
Normal freshly isolated CBA/J peritoneal cells as a source of B-1 cells (20 ± 4% of total), and harvested syngeneic LMNC from mice contact sensitized with TNP-Cl or injected with LPS (100 μg) i.p., both 1-h previously, were incubated together (5 × 106 peritoneal cells with 1 × 106 LMNC) with TNP-BSA Ag (50 μg/ml) for 60 min at 37°C. Then after washing with sterile pyrogen-free PBS three times, the cell mixture was transferred i.v. to predominantly B-1 cell-deficient recipients (CBA/N-xid) that were contact sensitized 3 days previously with TNP-Cl. Then 24-h later, recipients were challenged on the ears with 0.4% TNP-Cl and then 2- and 24-h ear swelling responses were determined.
Measurement of IL-4 in the supernatants of LPS-stimulated NKT cells
LMNC from wild-type and Jα18−/− mice were stimulated at 105 cells/ml with LPS (1 μg/ml). Twenty-four hours later, supernatants were collected and IL-4 was measured by ELISA. Briefly, plates were coated overnight with 2 μg/ml mAb 11B11 and blocked for 1 h with PBS containing 1% BSA. Then samples were diluted and incubated for 2 h at room temperature. After washing with PBS 0.1% Tween 20, biotinylated BVD6 anti-IL-4 mAb was added at 0.5 μg/ml for 1 h. The plates were then washed again and treated for 1 h with streptavidin-peroxidase (Amersham Biosciences) at room temperature. Following addition of the substrate o-phenylenediamine dihydrochloride (Sigma-Aldrich), reaction was stopped with H2SO4 and absorbance was measured at 490 nm with a reference filter of 630 nm. Samples were tested in duplicate and IL-4 concentrations were expressed as nanograms per milliliter, calculated according to calibration curves established with serial dilutions of murine rIL-4 (R&D Systems).
Statistics were performed using the paired two-tailed Student t test, and p < 0.05 was taken as the level of significance.
LPS induces B-1 cell anti-hapten IgM Ab responses
Mice were treated with LPS and 1 day later their ears were challenged topically with the hapten Ag TNP. Then 1 day later, LPS vs Ag-induced early 2-h ear swelling responses were compared with those induced by cutaneous contact skin sensitization with the specific Ag TNP.
Furthermore, we compared responses induced by LPS in CBA/J mice to responses in male CBA/N-xid that lack B-1 cells, and thus do not mount early IgM responses that mediate ear swelling (17). LPS induced significant 2-h ear swelling responsiveness to TNP in CBA/J mice injected i.v. or i.p. or applied epicutaneously (Fig. 1,a, left, groups C-E), compared with negative controls just ear challenged with TNP (group A). Responses were similar to mice contact immunized with TNP, the specific Ag (group B). In contrast, identically LPS-treated or TNP-sensitized xid mice had responses equivalent to negative controls (Fig. 1 a, right, group A vs groups B-E). Responses began at 0.1–1.0 μg of LPS/mouse i.p. and were optimal at 10–100 μg/mouse (data not shown). We concluded that LPS rapidly induced 2-h ear swelling to challenge with the hapten that resembled those induced by contact immunization with the TNP Ag and likely were mediated by B-1 cells.
An ELISPOT assay showed that LPS injection induced TNP-specific IgM Ab-producing cells in the spleen, which were polyclonal in just 1 day postimmunization (Fig. 1,b, right, group B). Thus, numbers of anti-TNP and anti-BSA IgM-producing cells in the spleen (Fig. 1 b, left, group B) were significantly greater than those in xid male mice (group D), also suggesting LPS-induced B-1 cell responses.
LPS induces migration of peritoneal B-1 cells to the spleen to produce IgM Abs
Contact skin sensitization with hapten activates specific B-1 cells in their normal location in the peritoneal cavity to migrate to the spleen within 1 h, resulting in splenic anti-TNP IgM Ab-producing B-1 cells (6 , 48). Similarly, LPS injection induced B-1 cells harvested from the peritoneal cavity at 1 h to migrate to the spleen to produce anti-TNP Abs by 1 day after i.p. transfer. (Fig. 1,c). Naive xid mice were used as recipients because they have nearly absent background IgM responses (Fig. 1,c). Also, any LPS carried over from the donors would not stimulate B-1 cells that were absent in recipients. Transfer of 1-h LPS-activated peritoneal cells resulted in significantly greater anti-TNP IgM-producing cells in the spleen (Fig. 1,c, right), compared with mice transferred with peritoneal cells of CBA/J mice injected with PBS as control (Fig. 1 c, left). Thus, LPS rapidly induced peritoneal B-1 cells to migrate to the spleen to generate systemic B-1 cell Ab responses.
The rapid LPS induced anti-hapten IgM response is prolonged
To examine the role of iNKT cells in BALB/c background mice deficient in iNKT cells, we first tested wild-type BALB/c mice. We compared the time course of LPS-induced ear swelling responses to contact sensitization with the TNP Ag, both elicited at 1 day (Fig. 1,d). Remarkably, LPS induced a prolonged TNP ear swelling response that also had a 24-h late phase equivalent to the 2-h swelling (Fig. 1,d). In comparison, contact sensitization with TNP Ag induced only the early response at 1 day that was present at 2 and 4 h, but absent by 24 h after testing, as found previously (8, 14). The stronger, more prolonged LPS-induced responses appeared to be dose-dependent since injection of 10 μg of LPS resulted in an equivalent early 2-h phase, but an absent late phase at 24 h (Fig. 1 d). We concluded that the prolonged ear swelling response to LPS likely was an indication of the greater strength of the polyclonal B-1 cell IgM responses.
Transfer experiments showed that the late 24-h responses induced by LPS at 1 day were not due to T cells, and probably were due to IgM Abs produced by B-1 cells. Thus, lymphoid cells from LPS-treated mice harvested at 1 day transferred the 2- and the 24-h responses, and this was not affected by in vitro treatment of the cells with anti-αβ-TCR or anti-γδ-TCR mAb plus complement (Fig. 1,e, groups D and E vs B). Furthermore, sera from CBA/J harvested at 1 day post-LPS treatment, but not from LPS-treated B-1 cell-deficient xid mice, transferred both the 2- and 24-h reactivity (Fig. 1 f, group D vs C).
The LPS-induced responses likely were part of polyclonal B-1 cell IgM Ab stimulation because similar 2-h ear swelling responses at 1 day postimmunization were elicited by challenge for the first time with other Ag, such as another hapten OX (Fig. 1,g, group D vs C), or intradermally with the protein keyhole limpet hemocyanin (KLH) (Fig. 1 g, group F vs E). Reactions were not nonspecific because they were not elicited, nor augmented by, challenge with either low or high doses of croton oil (groups I and J and groups G and H), a proinflammatory but nonimmunogenic contact agent. Thus, LPS responses were polyclonal for Ag, but were not nonspecific.
Rapid LPS activation of the anti-hapten IgM response is CD1d restricted
In contact sensitization, rapid B-1 cell IgM Ab responses to TNP are caused by dual activation of the B-1 cells by specific Ag and IL-4 produced by CD1d-restricted iNKT cells (Ref. 22, and R. A. Campos, M. Szczepanik, A. Itakura, M. C. Leite-de-Moraes, M. Kronenberg, and P. W. Askenase, submitted for publication). Thus, we compared TNP-induced 2 h ear swelling of CS in CD1d−/− mice to responses induced by LPS vs wild-type controls. As expected, the TNP-specific 2-h ear swelling responses in 1 day TNP contact-sensitized wild-type mice were significantly decreased in CD1d−/− mice (Fig. 2,a, group A vs B). Similarly, at 1 day post-LPS injection, 2- and 24-h ear swelling responses to TNP challenge were strongly inhibited in CD1d−/− mice (Fig. 2 a, group D vs C), and thus were CD1d dependent.
The LPS-induced responses are dependent on iNKT cells
LPS-induced 1-day ear swelling was absent in Jα18−/− mice deficient in iNKT cells compared with BALB/c controls (Fig. 2,b, group B vs group D). The ELISPOT assay was confirmatory, because polyclonal increases of anti-BSA and anti-TNP IgM-producing cells in the spleen at 1 day following LPS injection in wild-type mice (Fig. 2 c, group B vs A) did not occur in Jα18−/− mice (group D vs C). The high background IgM-producing cells in the nonimmune Jα18−/− mice (group C) may be related to increased numbers of B-1 cells in these mice (17). We concluded that CD1d-restricted LPS-induced polyclonal B-1 cell IgM Ab responses depend on iNKT cells.
Early LPS-induced TNP ear swelling responses depend on IL-4
In CS, IL-4 is rapidly produced by activated iNKT cells and is required with Ag to coactivate the B-1 cells to rapidly produce IgM Abs (22 , and R. A. Campos, M. Szczepanik, A. Itakura, M. C. Leite-de-Moraes, M. Kronenberg, and P. W. Askenase, submitted for publication). Thus, injection of anti-IL-4 at skin immunization with TNP significantly decreased 2-h responses to TNP ear challenge at 1 day (Fig. 3,a, group B vs A and C). Importantly, LPS induced 2 h TNP ear swelling responses also were inhibited by anti-IL-4, and not by treatment with isotype control (Fig. 3 a, group E vs F and D). We concluded that polyclonal B-1 cell IgM responses induced by LPS were dependent on CD1d-restricted iNKT cells and IL-4 production, similar to early TNP immune responses induced by contact sensitization with the TNP Ag (Ref.22 , and R. A. Campos, M. Szczepanik, A. Itakura, M. C. Leite-de-Moraes, M. Kronenberg, and P. W. Askenase, submitted for publication).
Injection of LPS induces rapid expression of IL-4 in iNKT cells
Flow cytometry tested whether the IL-4 was produced by iNKT cells. iNKT cells among the LMNC double stained with allophycocyanin-CD1d-αGalCer tetramers and FITC-anti-TCRβ were analyzed for intracellular cytokines after LPS injection. Intracellular IL-4 was rapidly induced in iNKT cells with an onset at 60 min in the CD4+ subset following LPS injection (2.3% vs control 0.7%); intracellular IL-4 peaked at 90 min (13.4% in CD4+ iNKT cells and 16.4% in CD4− iNKT cells; Fig. 3,b, fourth panel, top and bottom row) and was gone at 120 min (data not shown). In contrast, LPS did not cause similar expression of IFN-γ in LMNC (Fig. 3,c) nor IL-4 in splenocytes (Fig. 3,e). Positive controls injected with α-GalCer (2 μg i.v.), a nonspecific activator of iNKT cells, had strong expression of both IL-4 (Fig. 3,b, second panel) and IFN-γ (Fig. 3 c, second panel). Thus, LPS injection induces rapid, preferential and transient production of IL-4 in hepatic iNKT cells, similar to contact sensitization with the specific Ag TNP (R. A. Campos, M. Szczepanik, A. Itakura, M. C. Leite-de-Moraes, M. Kronenberg, and P. W. Askenase, submitted for publication).
TLR4-negative and MyD88−/− mice have deficient iNKT cell-B-1 cell IgM responses
We determined whether the TLR4/MyD88 (23) pathway is involved in the LPS-induced iNKT cell and B-1 cell collaboration leading to early IgM responses. Injection of LPS into BALB/c mice with functional TLR4 elicited 2- and 24-h TNP ear swelling responses, (Fig. 4,a, group B vs A), while LPS unresponsive TLR4 dominant-negative mice did not (group D vs C). Similarly, LPS injection failed to elicit TNP ear swelling responses in C57BL/10ScNJ mice that have a deletion of the chromosome portion encoding TLR4 (Ref.24 ; Fig. 4 b, group B vs D).
Furthermore, LPS-induced TNP ear swelling at 2 and 24 h was absent in MyD88−/− mice that are deficient in the adaptor protein required for most signaling via TLR4 compared with positive responses in wild-type controls (Fig. 4,c, group B vs E), as were the splenic ELISPOT anti-TNP IgM responses to TNP at 1 day (Fig. 4,d). Importantly, TNP contact-sensitized MyD88−/− mice had intact 2-h ear swelling responses to TNP at 1 day (Fig. 4 c, group F). This suggests that MyD88-mediated signaling is not involved in these classical Ag-specific early responses in contrast to those induced by LPS. Thus, LPS-induced anti-TNP IgM B-1 cell responses that require IL-4 produced by CD1d-restricted iNKT cells are dependent on TLR4 and the MyD88 pathway.
Early anti-TNP responses induced by LPS do not depend on IL-12
iNKT cells have IL-12Rs, and in some instances LPS can stimulate iNKT cells indirectly via stimulating TLR4 of DC that produce IL-12 to then activate iNKT cells (24). IL-12 is thought to promote pre-existing interactions between Vα14 TCR of iNKT cells and postulated endogenous glycolipids (25) presented by CD1d on the DC. This seems to be the case in the indirect activation of iNKT cell IFN-γ release, and without any IL-4, on day 1 after LPS injection (26).
Thus, we investigated the role of IL-12 in the LPS-induced very early IL-4-dependent B-1 cell IgM responses. We compared the effect of treatment with anti-IL-12 vs anti-IL-4 on the LPS-induced 1-day ear swelling responses to TNP challenge. As before, anti-IL-4 treatment before LPS injection inhibited TNP ear swelling at 1 day, now shown at both 2 and 24 h (Fig. 5,a, group C vs D), compared with the isotype control. In contrast, similar treatment with anti-IL-12 did not inhibit the LPS response (group E vs F). Furthermore, IL-12−/− mice elicited LPS-induced 2- and 24-h responses at 1 day equivalent to BALB/c controls (Fig. 5 b, group F vs E).
Finally, ELISPOT assay showed that the anti-TNP IgM-producing spleen cell responses were not impaired in IL-12−/− mice (Fig. 5,c, group D vs C, left and middle) compared with high background responses in wild-type controls (Fig. 5 c, group B vs A). We concluded that the early LPS-induced iNKT cell and polyclonal B-1 cell collaboration did not depend on IL-12.
Flow cytometry demonstration of TLR4 in iNKT cells
The experiments above suggested that LPS could stimulate TLR4 of iNKT cells. We sought direct evidence that TLR4 are expressed by iNKT cells using flow cytometry using a mAb (MTS510) that binds the complex of TLR4 and MD2, an accessory molecule involved in TLR4 signaling (27). This mAb directly and strongly stained control thioglycolate-stimulated peritoneal macrophages (Fig. 6,a), but iNKT cells only were stained after permeabilization. Fig. 6,b shows that 19.1% of the CD1d/α-GalCer+, TCRαβ+, and CD4+ triple-positive subpopulation of splenocytes were TLR4 positive, compared with 0.8% of conventional tetramer-negative TCRαβ+ CD4+ cells. Simultaneous isotype controls for the anti-TLR4/MD2 mAb, respectively, stained 1.7 and 0.2% of these populations (Fig. 6 b). We concluded that murine iNKT cells express determinants of TLR4.
LPS stimulates iNKT cells to activate B-1 cells ex vivo to initiate T cell recruitment in vivo in CS
We determined whether the TLR4 of iNKT cells are functional. Thus, we tested whether LPS injection might lead to in vivo activation of iNKT cells to collaborate ex vivo with B-1 cells for eventual T cell recruitment. The assay we used was adoptive transfer of naive B-1 cells coactivated in vitro by TNP-BSA together with in vivo-stimulated iNKT cells as a source of IL-4. The cell mixture then was transferred into 3-day TNP-Cl immune xid mice tested for elicitation of CS. These immunized xid recipients generate CS-effector T cells but, because of absent B-1 cells, cannot recruit the T cells and thus have no 2-h and weak 24-h ear swelling CS responses to TNP challenge (Fig. 7, group B) (14).
As a positive control, naive B-1 cells were incubated in vitro for 1 h with TNP-BSA together with LMNC (35% iNKT cells) from donors contact sensitized with TNP 1 h previously that were able to fully reconstitute the CS responses (Fig. 7,a, group C). Importantly, LMNC harvested 1 h after LPS injection of wild-type mice had a similar ability (Fig. 7 a, group D), while LMNC from mice injected with LPS 1 h previously, but not incubated and transferred with Ag-stimulated B-1 cells, were inactive (group E). Negative controls were nonimmune xid mice just ear challenged with TNP (group A). Therefore, within 1 h, LPS injected in vivo induced the ability of liver iNKT cells to collaborate in vitro with naive B-1 cells stimulated with Ag to likely generate anti-TNP IgM Abs that led to recruitment of CS-effector T cells.
In vitro LPS activation of LMNC to collaborate with B-1 cells
We attempted to use LPS to activate iNKT cells in vitro. The LPS-stimulated LMNC were from B-1 cell-deficient H-2k xid mice. This eliminated LPS stimulation of any B-1 cells present in the LMNC. Furthermore, the B-1 cell-containing peritoneal cells stimulated with TNP-BSA Ag were from H-2k C3H/HeJ mice with defective TLR-4 responses to omit possible stimulation by any LPS contamination of the TNP-BSA or via LPS carried over from the in vitro incubation with LMNC.
The separately in vitro-stimulated populations were washed, combined, and incubated together for another 40 min at 37°C to allow for postulated LPS-induced iNKT cell release of IL-4 to activate the B-1 cells with Ag. After washing again, the cell mixture was transferred i.p. Recipients were nonimmune LPS-insensitive C3H/HeJ mice to avoid any effects of LPS carried over from the in vitro incubations. The TNP-BSA-stimulated peritoneal C3H/HeJ cells coincubated with the in vitro LPS-stimulated xid LMNC mediated significant TNP ear swelling responses in nonimmune C3H/HeJ mice, compared with controls just challenged with TNP (Fig. 7,b, group B vs control group A). In contrast, the separately incubated B-1 cell and iNKT cell populations did not (groups C and D). Transferred responses were positive at 2 h and in a late phase at 24 h (Fig. 7,b). These dual late responses were similar to those of mice injected with LPS (Fig. 1,d), or receiving 1-day immune cells (Fig. 1,e), or serum (Fig. 1 f) from LPS-injected donors.
Flow cytometry experiments confirmed that LPS treatment in vitro induced rapid expression of IL-4 but not IFN-γ in the CD4+ hepatic iNKT cells gated via expression of TCRαβ and CD1d/α-GalCer tetramers (Fig. 7,c). Thus, 2-h LPS incubation caused a dose response (1–100 μg/ml) increased expression of IL-4 but not IFN-γ in CD4+ hepatic iNKT cells compared with medium and isotype controls, while the positive control α-GalCer caused expression of both (Fig. 7 c).
We also performed in vitro experiments in which LPS-induced production of IL-4 by iNKT cells was measured by ELISA. Stimulation of wild-type LMNC with 1 μg/ml LPS induced IL-4 secretion in the supernatant (Fig. 7,d), while identical stimulation of LMNC of Jα18−/− mice induced no production of IL-4 (Fig. 7 d). These results suggest that LPS acted in vitro to stimulate the iNKT cells in the LMNC to produce IL-4 to coactivate the B-1 cell IgM responses. We conclude that LPS likely can stimulate iNKT cells in vitro to activate peritoneal B-1 cells to produce anti-TNP IgM Abs in the recipients by 1 day.
Determination that TLR4 and IL-4 expression by iNKT cells are needed for LPS-induced polyclonal B-1 cell responses
We used wild-type LMNC as a source of iNKT cells to reconstitute the defective LPS responses of Jα18−/− mice (Fig. 2, b and c). One day before LPS injection of groups of Jα18−/− mice, we compared i.p. transfer of naive iNKT cell-containing LMNC from either wild-type BALB/c controls (Fig. 8 a, group C) to TLR4-negative BALB/c background mice (group D). The iNKT cell-containing LMNC were transferred at a ratio of one donor per one recipient, or ∼1–2 × 106 LMNC, as previously described (16). After 24 h, the Jα18−/− recipients were treated with LPS and 1 day later, early 2- and 24-h LPS-induced ear swelling and splenic ELISPOT IgM responses were determined.
Wild-type LMNC reconstituted the swelling responses of LPS-treated Jα18−/− mice (Fig. 8 a, group C). In contrast, LMNC cells from the TLR4-defective donors that have LPS-unresponsive iNKT cells failed (group D). Because the Jα18−/− recipients are only deficient in iNKT cells, and have fully intact TLR4 on various cells, and are reconstituted with wild-type LMNC but not TLR4-negative LMNC, this suggests that TLR4 on iNKT cells are needed for the LPS responses. Furthermore, LMNC from IL-4−/− mice also failed to reconstitute LPS responses in Jα18−/− mice (group E), confirming that LPS activated TLR4 of iNKT cells for the production of IL-4. In contrast, transfer of LMNC from IFN-γ−/− mice restored responses (group F) similar to wild-type LMNC (group C), indicating that IFN-γ was not involved. Negative controls were noninjected Jα18−/− mice that just were ear tested with TNP (group A), and similarly ear-challenged Jα18−/− mice transferred with wild-type LMNC, but not injected with LPS (group G). These findings suggest the involvement of functional TLR4 of the iNKT cells.
An ELISPOT assay confirmed these findings. Jα18−/− mice reconstituted with wild-type LMNC and then injected with LPS had early anti-TNP IgM-producing cell responses in the spleen at 1 day compared with LPS-injected Jα18−/− that received no transfer, and LMNC from TLR4-negative donors did not reconstitute IgM Ab responses in the LPS-injected Jα18−/− mice, nor did LMNC from IL-4−/− mice (data not shown).
Other TLR agonists stimulate B-1 cells via iNKT cell activation
We determined whether other TLR agonists could stimulate iNKT cell and B-1 cell collaboration. Lipid A, the TLR4-binding component of LPS (28), like LPS rapidly induced 2- and 24-h TNP-elicited ear swelling in CBA mice (Fig. 9 a, group C). The TLR2 agonists peptidoglycan and zymosan (3) (groups D and E) and the TLR9 agonist CpG (3) also induced 2- and 24-h ear swelling responses (group F) (3), while the TLR3 stimulator dsRNA (29) did not (group G).
Thus, TLR2, TLR4 and TLR9 may be involved in rapid B-1 cell responses. To test for involvement of iNKT cells we similarly tested Jα18−/− vs BALB/c control mice. Lipid A and peptidoglycan responses were absent in Jα18−/− mice compared with wild-type controls (Fig. 9 b, group C vs D, and E vs F). This was not true of mice injected with CpG (group G vs H). We concluded that collaboration between iNKT cells and B-1 cells for rapid IgM responses can depend on TLR4 or TLR2, but that the TLR9 ligand CpG likely stimulates the B-1 cell responses independently of iNKT cells.
The principal finding of this study is that there are functional TLR4 on iNKT cells that can activate collaboration between innate-like iNKT cells and B-1 B cells. This is required for elicitation of acquired T cell effector immunity. LPS treatment in vivo and in vitro stimulated very rapid and preferential production of IL-4 in liver iNKT cells to coactivate the B-1 cells to produce polyclonal IgM Abs. The lipid A component of LPS and a TLR2 stimulant peptidoglycan induced similar iNKT cell-dependent B-1 cell responses, while the TLR9 activator CpG did not. A major implication of these findings is that microbial TLR ligands can stimulate very early polyclonal Ab responses that can allow for recruitment into the tissues of Ag-specific effector T cells, for subsequent local activation by APC.
We showed previously that B-1 cells are activated very early after Ag immunization to produce specific IgM Abs. These mediate a T cell-recruiting initiation phase that is separate from and is required for the elicitation of the late classical effector T cell phase of CS (17) and DTH (8). Demonstration that B-1 cells are involved in an innate immune-initiating cascade required to elicit CS and DTH led us to make a connection between TLR and the elicitation in vivo of acquired T cell immunity. We postulated that polyclonal stimulation of B-1 cell responses by the TLR4 ligand LPS could lead to production of anti-hapten IgM Abs that function to initiate the elicitation of CS. We hypothesized that the polyclonal-stimulated Abs could mediate recruitment of CS-effector T cells of appropriate Ag specificity, when such circulating cells were available, and their Ag was expressed by local APC.
One possible implication of these findings involves the fact that microbial TLR ligands like LPS can polyclonally stimulate B-1 cell, or possibly B-2 cell production of Abs against autoantigens. These Abs may then facilitate recruitment of autoantigen-specific effector T cells and thus contribute to the mediation of local autoimmune inflammation. Indeed, we have demonstrated LPS-induced polyclonal B-1 cell IgM responses against collagen type II (data not shown) that might be involved in the recruitment of anti-collagen-specific T cells in arthritis.
It has been known for a long time that LPS, a classical TLR4 ligand, can polyclonally stimulate Ab production by B cells, including B-1 cells (30, 31, 32, 33, 34). From the point of view of resistance to pathogens, rapid polyclonal Ab responses may be useful to having diverse Ab specificities available early after infection to aid in the recruitment of Ag-specific effector T cells, when they are subsequently available. This would enable T cell responses to microbial Ag peptides expressed on tissue APC, and thus allow Ag-specific local production of cytokines to mediate cellular immunity.
As expected from prior findings, our flow cytometry studies identified TLR4 on B-1 cells (data not shown). In contrast, TLR have not been associated with T cells until recently. Some γδ-T cells (35), and CD4+ CD25+ suppressor T cells (36, 37), can express TLR4. A new finding of this study is demonstration that TLR4 are present and are functional on iNKT cells. The presence of TLR2 and TLR4 on iNKT cells was suggested previously by experiments using PCR analysis of sorted double-stained NK1.1+ and TCRβ+ cells in B6 mice (38, 39). In this study, we showed that TLR4 are expressed in iNKT cells by flow cytometry using permeabilized cells, while macrophages could be stained directly. This is consistent with the recent demonstration that TLR4 play a role in intracellular signaling in some cells (40). We show that LPS acting via TLR4 and MyD88 can stimulate iNKT cell production of IL-4. The IL-4 is needed to mediate a rapid helper effect for the TLR4-stimulated production of IgM Abs by B-1 cells. Also, B-1 cell IgM Ab responses were activated when LPS was injected either i.p. or i.v. or given by epicutaneous application. The early IgM responses resulting from collaboration between the innate-like iNKT cells and B-1 cells can lead to the innate initiating cascade to elicit CS (17, 22). This innate cascade consists of complement activation of mast cells and platelets to produce vasoactive mediators (7, 8, 9, 10, 11, 12, 13) that stimulate local endothelial cells to permit recruitment into the tissues of effector T cells to elicit acquired immunity (14, 15, 16).
LPS-mediated B-1 cell and iNKT cell collaboration via IL-4 production was determined in several ways. First, LPS-induced responses were absent in B-1 cell-deficient xid mice, and in iNKT cell-deficient CD1d−/− and Jα18−/− mice. Second, collaboration was not present in TLR4-negative nor in MyD88−/− mice, that, respectively, are without either functional LPS receptors or the MyD88 adapter protein needed for intracellular signaling from TLR4. Third, anti-IL-4 treatment abolished this collaboration. Fourth, flow cytometry using specific iNKT cell tetramers demonstrated very rapid and preferential expression of IL-4 in hepatic iNKT cells within minutes postinjection of LPS. Fifth, rapid LPS activation of the iNKT and B-1 cell collaboration induced B-1 cell migration, after just 1 h, from their normal location in the peritoneal cavity to the spleen, to produce the polyclonal IgM Abs present in the serum after only 1 day.
Sixth, iNKT cells in LMNC, harvested from mice injected with LPS just 1 h previously, collaborated in vitro with Ag-stimulated B-1 cells to lead to the initiation of CS responses. Seventh, LPS-activated the iNKT cells in vitro to stimulate the B-1 cells to enable their adoptive transfer of 2- and 24-h ear swelling responses. Importantly, this was in a system in which LPS could not affect the B-1 cells that instead were stimulated by Ag and not by the LPS because C3H/HeJ B-1 cells were used. Also, LPS could not activate any B-1 cells in the LMNC in vitro, because LMNC from xid donors were used. Finally, LPS could not affect the recipients if carried over, because TLR4 dominant-negative mice were the recipients. Eighth, Jα18−/− iNKT cell-deficient mice with defective responses to LPS were reconstituted by transfer, before LPS injection, of wild-type LMNC but not by Jα18−/− LMNC nor LMNC from IL-4−/− donors, nor from TLR4-negative donors, while LMNC from wild-type and IFN-γ−/− mice succeeded.
Taken together, these findings suggest that LPS acting via TLR4 and MyD88 can directly and rapidly activate hepatic iNKT cells to release IL-4 to allow Ag stimulation of B-1 cell production of IgM Abs. Whether the resulting IL-4 production by iNKT cells also depends on endogenous glycolipid ligands of their Vα14+ TCR, like iGb3 (41), or on cytokines other than IL-12 produced by accessory cells (28) remains to be determined. The LPS-induced iNKT cell-dependent IgM Abs then can act to initiate the local recruitment of Ag-specific effector T cells to mediate the classical acquired immune CS response in vivo.
It is established that iNKT cells readily produce both IL-4 and IFN-γ. Stimulation by α-GalCer leads to early production of both cytokines. However, LPS-induced rapid and small but significant expression of IL-4 preferentially in hepatic iNKT cells, with no comparable early production of IFN-γ. In contrast, it was shown that at later times there is IFN-γ production without IL-4 (26). Early production of IL-4 without IFN-γ also occurs after immunization by cutaneous contact sensitization (R. A. Campos, M. Szczepanik, A. Itakura, M. C. Leite-de-Moraes, M. Kronenberg, and P. W. Askenase, submitted for publication). These differences may arise because of different signaling pathways for LPS or Ag stimulation, compared with α-GalCer.
This collaboration between TLR-stimulated innate-like iNKT cells and B-1 cells to produce Abs that can initiate local T cell recruitment may be important in other forms of cellular immunity besides CS and DTH. For instance, autoimmune disorders like arthritis are thought to be due to autoreactive T cells with specificity for peptides of joint constituents like those of collagen. The autoreactive T cells are thought to be generated from a breakdown of self tolerance (42). This is postulated due to microbial-derived PAMPs like LPS acting on TLR, enabling a breakdown of self tolerance (43). However, for these generated autoreactive effector T cells to be pathogenic in autoimmunity, they likely in addition require recruitment into the target tissues to then be able to interact with autoantigen expressed on local APC to allow elicitation of the local autoimmune inflammatory response. Abs may be required to recruit the effector T cells into the tissues. This would be one explanation for the synergy between Ab and T cell responses in arthritis (44), and also for the success of B cell depletion in arthritis treatment (45). An iNKT cell dependence of these Ab responses, and possible activation of iNKT cells to down-regulate their TCR, could explain the relative deficiency of iNKT cells in autoimmune diseases (46), particularly rheumatoid arthritis (47).
Similar to microbial PAMPs generating the autoreactive T cells, the same or other subsequent microbial infections may generate polyclonal Ab responses to serve such an Ab-mediated initiating function, perhaps to mediate relapses of arthritis. Generated Abs produced with specificity for self determinants present in the joints, such as collagen, can then recruit the anti-collagen effector T cells to mediate local autoimmune inflammation. Thus, these required separate steps; i.e., generation of polyclonal Abs and stimulation of autoreactive T cells could both be induced by microbes, perhaps even by different agents. TLR activation of APC induced by one infection may lead to a breakdown of T cell tolerance, while other microbes may provide TLR ligands to stimulate polyclonal Abs needed to recruit the autoreactive T cells. Therefore, treatments that aim at blocking the collaboration between iNKT cells and B-1 cells, or the effects of the recruiting Abs, such as therapies directed at complement and/or mast cells or platelets could synergize with existing therapies directed at the autoreactive T cells to produce better results in the treatment of such autoimmune processes.
We are indebted to Marilyn Avallone for secretarial and administrative skills and to Dr. Ruslan Medzitov for critical comments on the manuscript.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by National Institutes of Health Grants AI-59801 and AI-07174 to P.W.A.; institute funds from CNRS and Fondation pour lar Recherche Medicalae (FRM) (Equipe FRM/Jeune Investigateur en Allergologie) to M.C.L.-d.-M.; and the Polish Committee of Scientific Research to M.S.
Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; DC, dendritic cell; CS, contact sensitivity; DTH, delayed-type hypersensitivity; iNKT, invariant Vα14+Jα18+ NKT; TNP, trinitrophenyl; PCl, picryl chloride; OX, oxazolone; LMNC, liver mononuclear cell; KLH, keyhole limpet hemocyanin.