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The Journal of Immunology, 2008, 180, 3882 -3888
Copyright © 2008 by The American Association of Immunologists, Inc.
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TLR Agonists Promote Marginal Zone B Cell Activation and Facilitate T-Dependent IgM Responses1

Anatoly V. Rubtsov*, Cristina L. Swanson*, Scott Troy{dagger}, Pamela Strauch*, Roberta Pelanda* and Raul M. Torres2,*

* Integrated Department of Immunology, University of Colorado Denver and National Jewish Medical and Research Center, Denver, CO 80206; and {dagger} West High School, Denver, CO 80204


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although IgM serves as a first barrier to Ag spreading, the cellular and molecular mechanisms following B lymphocyte activation that lead to IgM secretion are not fully understood. By virtue of their anatomical location, marginal zone (MZ) B cells rapidly generate Ag-specific IgM in response to blood-borne pathogens and play an important role in the protection against these potentially harmful Ags. In this study, we have explored the contribution of TLR agonists to MZ B cell activation and mobilization as well as their ability to promote primary IgM responses in a mouse model. We demonstrate that diverse TLR agonists stimulate MZ B cells to become activated and leave the MZ through pathways that are differentially dependent on MyD88 and IFN-{alpha}β receptor signaling. Furthermore, in vivo stimulation of MZ B cells with TLR agonists led to a reduction in the expression of the sphingosine-1-phosphate (S1P) receptors expressed by MZ B cells and/or increased CD69 cell surface levels. Importantly, as adjuvants for a T cell-dependent protein Ag, TLR agonists were found to accelerate the kinetics but not magnitude of the Ag-specific IgM response. Together, these data demonstrate that in vivo TLR agonist treatment enhances the early production of Ag-specific IgM and activates MZ B cells to promote their relocation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The immune system recognizes invading pathogens using both nonspecific and specific mechanisms. The nonspecific component, innate immunity, recognizes pathogen-associated molecular patterns with a set of germline-encoded pattern-recognition receptors that are referred to as TLRs (1, 2, 3). In contrast, adaptive immunity identifies pathogens via highly specific Ag receptors expressed on the surface of T and B lymphocytes. The humoral immune response is a component of adaptive immunity in which Abs produced by B cells neutralize and aid in the clearance of extracellular microorganisms and prevent the spread of infections.

The primary Ab response to foreign Ags is characterized by the rapid production of low-affinity IgM (4, 5) followed by the production of other classes of Abs with higher affinity. The Ab response to a soluble protein Ag is weak to negligible but can be enhanced through the use of adjuvants, typically aluminum salts (alum), acting through poorly defined mechanisms. LPS is a component of Gram-negative bacteria and a ligand for TLR4 that has been long known to enhance mouse B cell Ab responses to both T-dependent protein and T-independent carbohydrate Ags (6, 7). This property has now been extended to other TLR ligands (8, 9) and it has been suggested that TLR signaling is obligate for optimal B cell Ab responses (10, 11, 12) although not required (13). Regardless, TLR agonists are clearly able to augment Ab responses in humans and mice (6, 7, 8, 9, 10, 11, 12) and is not surprising given that bacterial and viral pathogens present combinations of protein and carbohydrate Ags to B cells along with ligands for TLRs. Thus, TLR ligands must be considered in the nature of Ag because they almost certainly contribute to the Ab response to bona fide bacterial and viral pathogens such as Streptococcus pneumoniae and HIV.

Marginal zone (MZ)3 B cells reside in the spleen between the white and red pulp and in close proximity to the marginal sinus. At this location, MZ B cells rapidly encounter blood-borne Ags (14) and have been characterized as a major source of the Ag-specific IgM to T-independent Ags (15, 16, 17), but also have been shown to contribute to the Ab response to T-dependent Ags (18, 19, 20, 21). Accordingly, this B cell subset is critical for Ab protection against bacterial and viral infections at relatively early stages of infection (15, 22, 23, 24).

Compared with follicular (FO) B cells, MZ B cells are more readily activated upon Ag or LPS stimulation and more rapidly differentiate into Ig-secreting cells (25, 26, 27). These properties endow MZ B cells with an important role in host defense by providing rapid IgM-mediated protection at the early stages of an immune response, taking place after the immediate innate response and before the delayed specific response of the adaptive immune system (16). How innate immunity promotes Ab responses remains to be clarified, although given the variety of TLRs expressed by B cells (10, 11, 28, 29, 30, 31) suggests that these receptors are important in this regard. Nevertheless, the mechanism(s) by which TLR signaling in vivo promotes Ab responses is incompletely understood.

In this study, we demonstrate that agonists to intracellular and extracellular TLRs activate MZ B cells in vivo and promote their release from the MZ as previously shown for LPS (32). Despite sharing this general feature, we find that only select TLR agonists act as adjuvants for T-dependent Ags by promoting IgM production at early stages of an immune response, whereas other TLR agonists are much less effective at facilitating Ab production. Finally, we demonstrate that all TLR agonists increase the magnitude of the IgM response to protein Ags in Arhgef1–/– mice, whose MZ B cells do not normally participate in the T-dependent Ab response when immunized with standard alum adjuvant (21).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 mice were bred and maintained within the National Jewish Biological Resource Center. Arhgef1–/– animals were generated in, and bred on, a C57BL/6 genetic background (21). Ifnar1–/– mice (33) on the C57BL/6 background were provided by Dr. L. Lenz (National Jewish Medical & Research Center, Denver, CO). MyD88–/– mice were generated by S. Akira (34) and bred onto the C57BL/6 genetic background in the National Jewish Medical & Research Center animal facility. Animals were used between 8 and 12 wk of age and all manipulations were performed in accordance with the Institutional Animal Care and Use Committee.

Immunizations and Ab measurement

C57BL/6 and Arhgef1–/– mice were immunized i.p. with 100 µg of (4-hydroxy-3-nitrophenyl)acetyl-coupled chicken {gamma}-globulin (NP-CG) (Biosearch Technologies, California) in either Alu-Gel-S (aluminum hydroxide (alum) in PBS; Crescent Chemical) or individual TLR agonists at the following concentration: 50 µg of Pam3Csk4 (N-palmitoyl(S)-[2,3-bis(palmitoyloxy)-(2, RS)-propyl]-Cys-Ser-Lys4) (InvivoGen), 100 µg of poly(I:C); InvivoGen), 50 µg of LPS (Escherichia coli O26; B6), 100 µg of 3M-012 (3M Pharmaceuticals, a gift from R. Kedl). Absolute concentrations of NP-specific IgM Abs were determined by a standard ELISA protocol and the B1–8µ mAb was used as a standard for NP-specific IgM (21).

In vitro TLR stimulation

Splenic B cells were purified by negative enrichment using CD43-conjugated microbeads (Miltenyi Biotec) and MZ (CD21highCD1dhigh) and FO (CD21intCD1dint) B cells isolated with a MoFlo sorter (DakoCytomation) to >95% purity. For in vitro IgM production, supernatant was measured by ELISA from isolated MZ and FO B cells after 7 days of culture with medium or medium with 2 µg/ml Pam3Csk4, 25 µg/ml poly(I:C), 10 µg/ml LPS, or 5 µg/ml 3M-012. In some experiments, CD21highCD1dhigh MZ and CD21intCD1dint FO B cells were sorted to >98% and stimulated in vitro for 24 h with each TLR agonist at the same concentrations as described above for flow cytometric analysis.

MZ B cell purification and RNA preparation

MZ B cells of TLR agonist-treated mice were isolated as described above and RNA was isolated using TRIzol reagent (Invitrogen Life Technologies), and trace amounts of DNA were removed using a DNA-free kit (Ambion). cDNA was prepared from 1 µg of total RNA using a SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen Life Technologies). Quantitative PCR amplification was performed using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen Life Technologies) and detected on an MJ Research DNA Engine Opticon 2 real-time PCR machine. Primers for quantitative PCR of S1P receptors were as previously described (32).

Immunofluorescence histology

Spleens were harvested and frozen at –80°C in OCT compound (EM Sciences). Tissues were cut into 5- to 7-µm sections and dried at room temperature overnight. Sections were rehydrated with PBS for 20 min and blocked for 30 min with PBS, 2% BSA, 0.05% Tween 20. Ab mixtures were added and incubated for 45 min followed by three 5-min washes with PBS. Sections were allowed to dry, mounted, and analyzed with Zeiss Axivert 200M microscope (3i Marianas System) using Slidebook 4.0 software (Intelligent Imaging Innovations).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TLR agonist in vivo treatment promotes MZ B cell relocation

In vivo treatment with LPS promotes MZ B cell migration from the MZ into splenic follicles (35, 36) and we questioned whether this was a unique feature of TLR4 signaling or a general property of TLRs expressed by MZ B cells (31, 37). To test this, we used four distinct TLR agonists whose receptors are expressed either on the plasma membrane (TLR2 and TLR4) or intracellularly (TLR3 and TLR7). These TLRs have been characterized to signal via either a MyD88-dependent (TLR2 and TLR7) or independent (TLR3) pathway or, for TLR4, capable of signaling via either MyD88-dependent and -independent pathways (38). Wild-type mice were treated in vivo with PBS or agonists for TLR1/2 (Pam3Csk4), TLR3 (poly(I:C)), TLR4 (LPS), or TLR7 (3M-012) and 18 h later the spleens of treated mice were examined histologically for the presence of B cells within the MZ. In PBS-treated animals, MZ B cells are found as IgMhighIgDlow (red) cells separated from IgMmedIgDhigh FO B cells by MOMA-1+ metallophilic macrophages (green) (Fig. 1A). However, MZ B cells were no longer present in the MZ of wild-type spleens 18 h after in vivo treatment with each of the individual TLR agonists as indicated by the loss of IgMhighIgDlow MZ B cells external to metallophilic macrophages (Fig. 1A). Thus, as previously shown for LPS, each TLR agonist tested similarly promoted the emigration of MZ B cells from the MZ.


Figure 1
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  		FIGURE 1. In vivo activation and release of MZ B cells by TLR agonists. A, Immunofluorescence histology of spleen sections from wild-type C57BL/6 mice 18 h after immunization with PBS, Pam3Csk4 (TLR1/2), poly(I:C) (TLR3), LPS (TLR4), or 3M-012 (TLR7). Sections were stained for MOMA-1 (green), IgD (blue), and IgM (red) and are representative of three experiments. MZ B cells are depicted as IgMhighIgDlow and appear as a red ring of cells exterior to MOMA-1+ (green) cells in PBS sections. B, MZ B cells were purified from naive and immunized C57BL/6 mice and quantitative PCR of respective S1P receptors was performed. The results are expressed as relative amount of mRNA normalized to hypoxanthine phosphoribosyl transferase. Bars represent mean (±SD) of three independent experiments. C, Representative dot plots of CD21 CD1d expression 18 h after in vivo treatment with PBS or indicated TLR agonists. MZ B cell gates used for D are shown. D, CD69 (top histograms) and CD86 (bottom histograms) expression on C57BL/6 MZ B cells (gated as CD21highCD1dhigh) 18 h after in vivo treatment with PBS or indicated TLR agonists. E, CD69 expression on MZ B cells (gated as CD21highCD1dhigh) from Ifnar1–/– mice treated for 18 h with PBS or poly(I:C). WT, Wild type.

 
In response to in vivo LPS treatment, MZ B cells have previously been shown to migrate into adjacent splenic follicles (35, 36). To determine whether MZ B cells remained present in the spleen after in vivo treatment with other TLR agonists, we examined splenocytes by flow cytometric analysis 18 h after treatment (Fig. 1C). These experiments revealed that MZ B cells were present in the spleen, although not in the MZ (Fig. 1A), after in vivo TLR agonist treatment, allowing us to evaluate their activation status (Fig. 1D). Together, these data suggest that a common feature of TLR signaling by MZ B cells is to promote their relocation from the MZ and migration into either an adjacent follicle or possibly red pulp.

In vivo modulation of MZ B cell S1P receptor expression by TLR agonists

The previous findings suggest that TLR agonists act generally in vivo to promote the release of MZ B cells from the MZ. B cell localization to the MZ requires expression of the S1P1 receptor (32), whereas MZ B cell retention is dependent on {alpha}Lβ2 (LFA-1) and {alpha}4β1 (VLA-4) integrins to ICAM-1 and VCAM-1, respectively, (36). Furthermore, TLR4 in vivo stimulation has previously been shown to facilitate MZ B cell emigration out of the MZ by reducing S1P1, S1P3, and S1P4 receptor expression and, consequently, the MZ B cell S1P response (32). Thus, we tested whether in vivo treatment with diverse TLR agonists also promoted reduced expression of MZ B cell S1P receptors. MZ B cells were sorted as CD21highCD1dhigh from mice stimulated with TLR agonists for 18 h and quantitative PCR was used to determine mRNA level of S1P1, S1P3, and S1P4 receptors. Importantly, in vivo TLR agonist treatment, or in vitro treatment of sorted MZ and FO B cells, did not significantly alter the expression of CD21 or CD1d on these B cell populations (Fig. 1C and data not shown), making it unlikely that these analyses were influenced by FO B cell contamination. As shown in Fig. 1B, in vivo treatment with TLR2, TLR4, and TLR7 agonists promoted a substantial reduction in the mRNA expression of all three S1P receptors. In contrast, the poly(I:C) TLR3 agonist did not alter the mRNA level of either the S1P1, S1P3, or S1P4 receptors.

The above results extend to MZ B cells the previous findings that in vivo poly(I:C) treatment only modestly reduces S1P1 receptor mRNA expression in total (unfractionated) splenic B cells (39). Nevertheless, poly(I:C) treatment is effective at inducing the release of MZ B cells (Fig. 1A) and has been shown to attenuate B cell S1P responsiveness by an alternate mechanism. Specifically, administration of poly(I:C) promotes increased expression of the CD69 activation marker that, in turn, inhibits S1P1 surface expression (39). Thus, we assessed how in vivo treatment with this TLR3 agonist compared with the other TLR agonists in ability to induce CD69 expression on MZ B cells. These results show that administration of poly(I:C) increased CD69 expression to a greater extent than the TLR2 agonist, but less than either the TLR4 or TLR7 agonists (Fig. 1D). Furthermore, the elevated expression of CD69 induced by poly(I:C) was entirely dependent on IFN-{alpha}β receptor signaling since CD69 surface expression was not increased on MZ B cells when IFN-{alpha}β receptor-1-deficient mice (Ifnar1–/–) were injected with this TLR3 agonist (Fig. 1E).

CD69 is an activation Ag indicating that in vivo TLR agonists induced the activation of MZ B cells. This was further confirmed by analysis of CD86 expression that is also increased upon B cell activation. As with CD69, all TLR agonists promoted increased expression of CD86 (Fig. 1D), confirming TLR agonists activate MZ B cells in vivo.

Together, these data demonstrate that in vivo TLR agonist treatment in general promotes the activation and efficient release of MZ B cells from the MZ. However, to diminish S1P responsiveness by MZ B cells, TLRs appear to differentially signal either down-regulation of S1P receptor mRNA expression and/or increased expression of CD69 and, presumably, subsequent diminished expression of the S1P1 receptor on the cell surface.

TLR agonists signal MZ B cell release by distinct pathways

TLRs transmit signals via adaptor molecules that have been characterized to proceed by two common pathways. The majority of TLRs, including TLR2, TLR4, and TLR7 rely on the MyD88 adaptor molecule, although TLR4 can also signal in a MyD88-independent manner (38). In contrast, TLR3 does not signal via MyD88 but instead depends on the TRIF adaptor (38). However, despite using these distinct signaling pathways, all TLR agonists tested efficiently induced the relocation of MZ B cells. This promoted us to assess the contribution of MyD88 in signaling the release of MZ B cells by the different TLR agonists. To accomplish this, MyD88–/– mice were treated with each of the TLR agonists and spleens were examined by histology (Fig. 2A). These results show that agonists to TLR2, TLR4, and TLR7 were ineffective at promoting the migration of MZ B cells out of the MZ in MyD88-deficient animals, whereas the TLR3 agonist was still capable of inducing MZ B cell release from the MZ. These data demonstrate that agonists to TLR2, TLR4, and TLR7 release MZ B cells from the MZ through a MyD88-dependent signaling pathway that results in reduced S1P receptor expression and presumably response.


Figure 2
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  		FIGURE 2. TLR agonists rely on different signaling pathways to release MZ B cells. A and B, MyD88–/– (top row) and Ifnar1–/– (bottom row) mice were treated with PBS or indicated TLR agonists and 18 h later spleen sections were examined by immunofluorescence histology as described in Fig. 1 and are representative of two to three experiments.

 
TLR3 is the only TLR characterized to signal in a strict MyD88-independent manner and, although poly(I:C) treatment was inefficient at altering mRNA expression of the S1P receptors, it did promote increased expression of surface CD69 expression in an IFN-{alpha}β-dependent manner (Fig. 1). To determine whether the IFN-{alpha}β receptor-1 signaling was also required for TLR-mediated MZ B cell release, we immunized Ifnar1–/– mice with TLR agonists (Fig. 2B). Histological sections from PBS-treated animals revealed Ifnar1–/– mice have MZs that are reduced in size compared with either wild-type or MyD88–/– animals. Nevertheless, injection of poly(I:C) failed to release MZ B cells in Ifnar1–/– mice, while TLR2, TLR4, and TLR7 agonists were all capable of promoting release of Ifnar1–/– MZ B cells. Therefore, in contrast to all other tested TLRs, TLR3 relies exclusively on increased CD69 surface expression that is sufficient to release MZ B cells from the MZ and presumably via promoting the decreased expression of S1P1 from the cell surface (39).

These results further underlie the existence of two distinct signaling pathways used by TLRs to not only activate MZ B cells but also to promote MZ B cell emigration from the MZ.

TLR agonists act as adjuvants in the T-dependent IgM Ab response

MZ B cells rapidly respond to foreign Ag exposure to produce Ag-specific IgM and we find that each TLR agonist tested in vivo activated and released MZ B cells from the MZ within the first 18 h after treatment (Fig. 1A). Although the requirement for TLR signaling in the T-dependent primary Ab response is arguable (10, 13), it is universally accepted that TLR agonists are able to augment Ab responses. Indeed, natural and synthetic agonists for TLRs have been shown to promote Ab production in vivo and in vitro (8, 40, 41, 42, 43, 44) and induce Ab production by both MZ and FO B cells (31, 37). Thus, we next addressed whether TLR agonists, when injected in combination with a T-dependent Ag and in the absence of alum, could promote an Ag-specific IgM Ab response.

Wild-type mice were immunized with the NP hapten coupled to chicken {gamma}-globulin (NP-CG) either in PBS, alum, or with individual TLR agonists and Ag-specific IgM Ab production was measured 4 and 7 days postimmunization. Immunization with NP-CG in alum alone induces a significant Ag-specific IgM response by 7 days that was typically 7- to 10-fold increased over preimmune levels or immunization of NP-CG in PBS (Fig. 3A). When compared with preimmune titers or immunization with Ag in PBS, each TLR agonist was observed to promote Ag-specific IgM 1 wk after immunization (Fig. 3A). However, the magnitude of the NP-specific IgM response promoted by TLR agonists 1 wk after immunization was in all cases reduced (TLR2 = 53%; TLR3 = 31%; TLR4 = 61%; and TLR7 = 21%) compared with that induced by NP-CG in alum. Total serum IgM was also measured at day 0 and 1 wk after immunization and revealed that LPS treatment promoted a 2-fold increase in total serum IgM levels by day 7 relative to day 0 and likely accounting for at least a proportion of the NP-specific IgM response (data not shown). None of the other TLR agonists induced elevated total serum IgM levels.


Figure 3
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  		FIGURE 3. TLR agonists accelerate the kinetics of the IgM Ab response to T-dependent Ags. A, NP-specific IgM in serum at day 0 (preimmune; open bars), day 4 (light gray bars), and day 7 (dark gray bars) after immunization of C57BL/6 mice with 0.1 mg of NP-CG in PBS, alum, or indicated TLR agonists. Symbols represent individual mice and results are representative of two experiments. B, MZ ({blacksquare}) and FO ({square}) B cells were isolated and cultured for 7 days in the presence of medium, 25 µg/ml poly(I:C), 10 µg/ml LPS, or 5 µg/ml 3M-012. IgM was subsequently measured in supernatant by ELISA. Data are representative of two independent experiments. nd, Not detected.

 
Despite promoting a significantly reduced Ag-specific IgM response at day 7 compared with alum, we found that agonists to TLR2, TL4, and TLR7 instead accelerated the kinetics of NP-specific IgM production. That is, the Ag-specific IgM response peaked at day 4 postimmunization when TLR2, TLR4, and TLR7 agonists were used as adjuvants and was ~3-, 10-, and 4-fold above the levels generated when alum was used as adjuvant, respectively (Fig. 3A).

Our above in vivo findings are consistent with recent in vitro studies showing that agonists to TLR2, TLR4, or TLR7 directly induce MZ and FO B cells to secrete Ab (31). However, the influence of in vitro TLR3 signaling by B cells was not reported in that study and we find that in vivo poly(I:C) TLR3 agonist treatment augments, albeit modestly, NP-specific IgM at day 7 (~2-fold over PBS). To directly test whether TLR3 signaling is able to induce IgM Ab production in vitro, MZ and FO B cells were sorted and cultured in the presence of the TLR3, TLR4, or TLR7 agonists. After 7 days of culture, supernatants were measured for IgM and revealed that, as previously reported (25, 26, 31), stimulation via TLR4 elicited significant amounts of IgM from MZ and FO B cells, although considerably enhanced in the former subset (Fig. 3B). TLR7 agonist treatment of purified B cells also resulted in considerable IgM production from MZ, but not FO, B cells, whereas TLR3 stimulation led to negligible IgM production from both populations. Thus, despite the ability of all TLR agonists (including TLR3) to efficiently induce increased expression of CD69 and migration of MZ B cells out of the MZ, a TLR3 agonist is not able to directly induce Ab secretion by B cells.

Taken together, these data demonstrate that TLR agonists are able to act as adjuvants to promote an Ag-specific IgM response to protein Ags. Importantly, as adjuvants, TLR2, TLR4, or TLR7 agonists function by accelerating the kinetics in which Ag-specific IgM is produced and present in serum, a feature that would clearly confer survival benefits to the host before high-affinity somatically mutated isotype-switched B cells could be generated.

TLR agonists rescue the T-dependent Arhgef1–/– IgM Ab response

Arhgef1 (Human Genome Organization nomenclature; formerly known as Lsc) is a signaling molecule that activates RhoA in response to G protein-coupled receptor signaling and we have previously shown that Arhgef1–/– MZ B cells do not contribute to the T-dependent IgM response despite responding normally to T-independent Ags (21). Thus, MZ B cells exhibit a differential requirement for Arhgef1 in mounting Ab responses that are dependent on the nature of Ag. Of interest, we have shown that Arhgef1 functions to regulate MZ B cell integrin adhesion and Arhgef1–/– MZ B cells inefficiently migrate out of the MZ after in vivo treatment with a suboptimal dose (17.5 µg) of LPS that is sufficient to completely release wild-type MZ B cells (21). Having shown that all tested TLR agonists promote the release of wild-type MZ B cells, we assessed whether Arhgef1–/– MZ B cells would migrate out of the MZ in response to in vivo treatment with optimal concentrations of LPS (50 µg) or other TLR agonists. Fig. 4A shows that, similar to wild-type, all TLR agonists promoted the efficient release of mutant MZ B cells, including LPS when injected at a dose of 50 µg. These data demonstrate that MZ B cells do not require Arhgef1 activity to be released from the MZ in response to optimal concentrations of TLR agonists.


Figure 4
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  		FIGURE 4. TLR agonists induce the release of Arhgef1–/– MZ B cells and enhance the Arhgef1–/– T-dependent IgM response. A, Arhgef1–/– mice were treated in vivo with PBS or the indicated TLR agonists and 18 h later B cells within the MZ were assessed by immunofluorescence histology of spleen sections. Staining is as described in Fig. 1. B, Arhgef1–/– mice ({blacksquare}) were immunized with 0.1 mg of NP-CG in alum alone (–) or together with 50 µg of Pam3Csk4 (TLR1/2), 100 µg of poly(I:C) (TLR3), 50 µg of LPS (TLR4), or 100 µg of 3M-012 (TLR7) and NP-specific IgM in serum 7 days later measured by ELISA. The NP-specific IgM response at day 7 in response to NP-CG in alum is shown for C57BL/6 mice by the open bar. Bars represent mean (±SEM) of at least six mice in each immunization.

 
Given that TLR agonists were capable of inducing the release of Arhgef1–/– MZ B cells from the MZ, we next asked whether TLR agonists could improve the T-dependent IgM response when included in the conventional immunization of NP-CG in alum. The results from these experiments (Fig. 4B) demonstrate that each of the TLR agonists was able to augment the Arhgef1–/– IgM response to this protein Ag with agonists to TLR2 or TLR4 eliciting an NP-specific IgM response comparable to that of wild-type 1 wk after immunization. Thus, these data show that in vivo TLR agonist treatment is sufficient to induce the release of Arhgef1-deficient MZ B cells and restore normal IgM response to T-dependent Ag in mutant mice.

We have previously suggested that different types of Ags (T dependent vs T independent) transmit distinct Ag receptor signals that control MZ B cell release through different pathways (21). These data demonstrate that TLR agonists are capable of activating and releasing MZ B cells in a manner independent of Arhgef1. Based on these and previous data, we proposed that the magnitude and strength of signals transmitted by T-dependent protein Ags along with TLR ligands determine the ability of Arhgef1-deficient MZ B cells to leave the MZ. The signal induced by a T-dependent protein Ag requires Arhgef1 activity to regulate appropriate adhesion and chemotaxis of MZ B cells. In contrast, TLR agonists induce either quantitatively or qualitatively distinct signals that do not depend on Arhgef1. In this regard, it remains possible that additional, ubiquitously expressed RGS-RhoGEF family members such as PDZ-RhoGEF or LARG (45, 46), may be used to regulate chemotaxis and adhesion. Regardless, TLR signaling augments the T-dependent IgM response in the absence of Arhgef1 and, in the future, it will be important to elucidate the signaling pathways initiated by different agents and the mechanisms used for regulating adhesion and chemotaxis by this important B cell population.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Innate and adaptive immune responses cooperate to provide effective immune protection. The recognition of pathogens by TLRs is important in this regard as illustrated by the ability of TLRs to signal dendritic cell maturation and subsequent T cell activation (47). B cells also express TLRs (31, 37) providing an additional mechanism in which these innate receptors can influence adaptive immunity. In this study, we have examined how TLR agonists act in vivo to influence MZ B cell activation and migration in addition to their ability to promote Ab responses to protein Ags. Our data demonstrate that TLR agonists appear to act generally to activate and mobilize MZ B cells and, when used as adjuvants, accelerate the kinetics but not the magnitude of the Ag-specific IgM Ab response. Clearly, this latter property of TLR signaling would have obvious importance in countering a pathogenic insult after the innate immune response but before the generation of high-affinity somatically mutated Abs.

MZ B cell localization within the MZ is dependent on their response to the S1P lysophospholipid and adhesion to integrin ligands (32, 36). In vivo treatment with either heat-killed E. coli or purified LPS has long been appreciated to promote the migration of B cells out of the MZ (35, 36, 48, 49) and Cyster and colleagues (32) have provided a mechanistic basis for this observation. Namely, upon LPS stimulation, MZ B cells reduce S1P receptor expression and response and consequently are guided into B cell follicles by the CXCR5 chemokine receptor responding to the CXCL13 B cell chemokine (32). Our data demonstrate that in addition to LPS acting on TLR4, agonists to TLR2, TLR3, and TL7 similarly lead to the rapid release of MZ B cells from the MZ, suggesting this is a general feature of TLR signaling by MZ B cells. MZ B cell release by TLR2, TLR4, and TLR7 signaling was dependent on the MyD88 adapter protein, whereas TLR3 signaling was MyD88 independent but IFNR1 dependent. These findings are in accord with the previously characterized dependence of signaling by these TLRs on this adaptor and cytokine receptor.

As with LPS in vivo treatment, administration of TLR agonists to TLR2 and TLR7 similarly promoted decreased transcript levels of all three of the S1P receptors expressed by MZ B cells. However, in vivo TLR3 agonist treatment did not significantly alter MZ B cell S1P receptor expression and similar to that previously shown for total splenic B cells (39). Instead, poly(I:C) induced increased expression of plasma membrane CD69 levels that has been shown to antagonize surface expression of the S1P1 receptor and S1P response (39). Because poly(I:C) efficiently promotes MZ B cell release without reducing S1P receptor mRNA expression, this suggests that a relatively modest increase in CD69 surface expression is sufficient to promote S1P1 receptor internalization, allowing MZ B cell release. Furthermore, the inability of poly(I:C), but not other TLR agonists, to up-regulate CD69 expression on Ifnar1–/– MZ B cells prevents their release providing further evidence that TLR3-mediated MZ B cell release relies exclusively on CD69 up-regulation. Taken together, our data would suggest that a general feature of TLR signaling in vivo by MZ B cells is to promote their release from the MZ although different TLRs rely on independent signaling pathways to effect this release. Our experiments show that in vivo TLR stimulation can activate MZ B cells and promote emigration out of the MZ. Whether in vivo administered TLR agonists act directly on MZ B cells to promote these events or act indirectly via accessory cells is not clear. In support of directly acting on MZ B cells, TLR agonist treatment of purified MZ B cells in vitro induces expression of the CD69 and CD86 activation Ags and reduces S1P receptor mRNA expression (data not shown). Furthermore, we find that in vivo treatment with flagellin, a ligand for TLR5 that is not expressed by MZ B cells (31, 37), does not promote MZ B cell release (data not shown).

MZ B cells not only make significant contributions to the T-independent Ab response (15), this B cell subpopulation also participates in the Ab response to T-dependent protein Ags (18, 19, 20, 21). Thus, TLR signaling in the context of an encounter with blood-borne pathogen would be expected to mobilize Ag-specific MZ B cells as they mount an appropriate Ab response. Indeed, LPS is well known to enhance the Ab response to T-dependent and T-independent Ags (6, 7). In this study, we have focused on the adjuvant activity of TLR agonists in the induction of the primary IgM response early after immunization and contrasts to previous reports evaluating the contribution of in vivo TLR signaling in the IgM response relatively late (7–12 days) (10, 13). We show that all TLR agonists tested act as adjuvants to promote an Ag-specific IgM response to a T-dependent Ag, although with variable efficiency. In particular, the poly(I:C) TLR3 agonist was the least efficient at promoting an Ab response to a protein Ag, whereas agonists to TLR2, TLR4, and TLR7 all led to significant Ag-specific IgM production compared with standard alum. One of the more important findings of this work is that these latter TLR agonists induce considerable Ag-specific IgM Ab production early in the response, by day 4, which was significantly elevated compared to alum alone. Thus, in contrast to the established kinetics of T-dependent Ab response described for protein Ags in alum (4, 50), these data indicate that bona fide pathogens would elicit the rapid production of Ab by virtue of simultaneously engaging a TLR. Furthermore, our findings also show that TLR signaling by MZ B cells in the absence of Ag receptor engagement also leads to emigration from the MZ and it will be of interest to determine how Ag-nonspecific MZ B cell migration promotes immune protection.

Finally, these data also address the role of Ag receptor and TLR signaling in the release of Arhgef1-deficient MZ B cells and induction of IgM production in Arhgef1-deficient mice. Previously, we have shown that Arhgef1 regulates MZ B cell adhesion and migration and suggested that different types of Ags (T dependent vs T independent) control MZ B cell release through different pathways (21). In this study, we demonstrate that TLR agonists are capable of activating and releasing Arhgef1-deficient MZ B cells. Based on these and previous data (21), we proposed that the magnitude and strength of signals transmitted by T-dependent Ags and TLR agonists determines the ability of Arhgef1-deficient MZ B cells to leave the MZ. The signals induced by T-dependent protein Ag require functional Arhgef1 to induce proper adhesion and chemotaxis of MZ B cells, whereas TLR agonists induce either quantitatively or qualitatively distinct signals that facilitate MZ B cell mobilization in the absence of Arhgef1. Consequently, TLR adjuvants facilitate an IgM response in the absence of Arhgef1 that is comparable to wild-type. It will be important to elucidate the signaling pathways initiated by different agents and the mechanisms used for regulating adhesion and chemotaxis.

It is important to note that our data do not formally demonstrate that MZ B cells are responsible for the accelerated IgM production in wild-type animals upon in vivo treatment with TLR agonists. However, the ability of TLR agonists to mobilize MZ B cells and the rapid kinetics with which Ag-specific IgM is generated would be consistent with this notion. Further evidence is also suggested by the ability of TLR agonists to both promote the release of mutant MZ B cells and restore the IgM response in Arhgef1–/– animals. Nevertheless, TLR agonists are also able to act on other cell types, such as dendritic cells, that in turn could also promote the early IgM response.

In summary, these data demonstrate that TLR agonists act in vivo to activate MZ B cells and promote their migration out of the MZ. Furthermore, when used as adjuvants with a protein Ag, TLR agonists are able to accelerate the kinetics, but not the magnitude, of the Ag-specific IgM response. Collectively, our results establish TLRs as potent activators of MZ B cells and, additionally, point toward their role in induction of rapid IgM response to foreign Ags. It will be of interest in the future to determine whether distinct TLRs signal differentially to MZ B cells imparting information about the nature of the offending Ag.


   Acknowledgments
 
We thank Ross Kedl for the gift of TLR agonists and critical comments on the experimental results, Josh Loomis for help with cell sorting, and members of the R&R laboratory for discussion.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by National Institutes of Health Grants AI052310 (to R.P.) and AI052157 (to R.T.) and an American Association of Immunologists John H. Wallace Summer Fellowship (to S.T.). Back

2 Address correspondence and reprint requests to Dr. Raul M. Torres, Integrated Department of Immunology, University of Colorado Denver and National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: torresr{at}njc.org Back

3 Abbreviations used in this paper: MZ, marginal zone; FO, follicular; S1P, sphingosine-1-phosphate; NP, (4-hydroxy-3-nitrophenyl)acetyl; CG, chicken {gamma}-globulin; Pam3Csk4, N-palmitoyl(S)-[2,3-bis(palmitoyloxy)-(2, RS)-propyl]-Cys-Ser-Lys4. Back

Received for publication October 11, 2007. Accepted for publication January 9, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Medzhitov, R., P. Preston-Hurlburt, C. A. Janeway, Jr. 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388: 394-397. [Medline]
  2. Iwasaki, A., R. Medzhitov. 2004. Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5: 987-995. [Medline]
  3. Beutler, B., Z. Jiang, P. Georgel, K. Crozat, B. Croker, S. Rutschmann, X. Du, K. Hoebe. 2006. Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large. Annu. Rev. Immunol. 24: 353-389. [Medline]
  4. Jacob, J., R. Kassir, G. Kelsoe. 1991. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl: I. The architecture and dynamics of responding cell populations. J. Exp. Med. 173: 1165-1175. [Abstract/Free Full Text]
  5. Liu, Y. J., J. Zhang, P. J. Lane, E. Y. Chan, I. C. MacLennan. 1991. Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens. [Published erratum appears in 1992 Eur. J. Immunol. 22: 615.]. Eur. J. Immunol. 21: 2951-2962. [Medline]
  6. Coutinho, A., E. Gronowicz, W. W. Bullock, G. Moller. 1974. Mechanism of thymus-independent immunocyte triggering: mitogenic activation of B cells results in specific immune responses. J. Exp. Med. 139: 74-92. [Abstract]
  7. Seppala, I. J., O. Makela. 1984. Adjuvant effect of bacterial LPS and/or alum precipitation in responses to polysaccharide and protein antigens. Immunology 53: 827-836. [Medline]
  8. Bernasconi, N. L., N. Onai, A. Lanzavecchia. 2003. A role for Toll-like receptors in acquired immunity: up-regulation of TLR9 by BCR triggering in naive B cells and constitutive expression in memory B cells. Blood 101: 4500-4504. [Abstract/Free Full Text]
  9. Krieg, A. M., A. K. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale, G. A. Koretzky, D. M. Klinman. 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374: 546-549. [Medline]
  10. Pasare, C., R. Medzhitov. 2005. Control of B-cell responses by Toll-like receptors. Nature 438: 364-368. [Medline]
  11. Sen, G., A. Q. Khan, Q. Chen, C. M. Snapper. 2005. In vivo humoral immune responses to isolated pneumococcal polysaccharides are dependent on the presence of associated TLR ligands. J. Immunol. 175: 3084-3091. [Abstract/Free Full Text]
  12. Ruprecht, C. R., A. Lanzavecchia. 2006. Toll-like receptor stimulation as a third signal required for activation of human naive B cells. Eur. J. Immunol. 36: 810-816. [Medline]
  13. Gavin, A. L., K. Hoebe, B. Duong, T. Ota, C. Martin, B. Beutler, D. Nemazee. 2006. Adjuvant-enhanced antibody responses in the absence of Toll-like receptor signaling. Science 314: 1936-1938. [Abstract/Free Full Text]
  14. Kraal, G.. 1992. Cells in the marginal zone of the spleen. Int. Rev. Cytol. 132: 31-74. [Medline]
  15. Martin, F., A. M. Oliver, J. F. Kearney. 2001. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 14: 617-629. [Medline]
  16. Martin, F., J. F. Kearney. 2002. Marginal-zone B cells. Nat. Rev. Immunol. 2: 323-335. [Medline]
  17. Pillai, S., A. Cariappa, S. T. Moran. 2005. Marginal zone B cells. Annu. Rev. Immunol. 23: 161-196. [Medline]
  18. Song, H., J. Cerny. 2003. Functional heterogeneity of marginal zone B cells revealed by their ability to generate both early antibody-forming cells and germinal centers with hypermutation and memory in response to a T-dependent antigen. J. Exp. Med. 198: 1923-1935. [Abstract/Free Full Text]
  19. Attanavanich, K., J. F. Kearney. 2004. Marginal zone, but not follicular B cells, are potent activators of naive CD4 T cells. J. Immunol. 172: 803-811. [Abstract/Free Full Text]
  20. Phan, T. G., S. Gardam, A. Basten, R. Brink. 2005. Altered migration, recruitment, and somatic hypermutation in the early response of marginal zone B cells to T cell-dependent antigen. J. Immunol. 174: 4567-4578. [Abstract/Free Full Text]
  21. Rubtsov, A., P. Strauch, A. Digiacomo, J. Hu, R. Pelanda, R. M. Torres. 2005. Lsc regulates marginal-zone B cell migration and adhesion and is required for the IgM T-dependent antibody response. Immunity 23: 527-538. [Medline]
  22. Wardemann, H., T. Boehm, N. Dear, R. Carsetti. 2002. B-1a B cells that link the innate and adaptive immune responses are lacking in the absence of the spleen. J. Exp. Med. 195: 771-780. [Abstract/Free Full Text]
  23. Zandvoort, A., W. Timens. 2002. The dual function of the splenic marginal zone: essential for initiation of anti-TI-2 responses but also vital in the general first-line defense against blood-borne antigens. Clin. Exp. Immunol. 130: 4-11. [Medline]
  24. Gatto, D., C. Ruedl, B. Odermatt, M. F. Bachmann. 2004. Rapid response of marginal zone B cells to viral particles. J. Immunol. 173: 4308-4316. [Abstract/Free Full Text]
  25. Snapper, C. M., H. Yamada, D. Smoot, R. Sneed, A. Lees, J. J. Mond. 1993. Comparative in vitro analysis of proliferation, Ig secretion, and Ig class switching by murine marginal zone and follicular B cells. J. Immunol. 150: 2737-2745. [Abstract]
  26. Oliver, A. M., F. Martin, G. L. Gartland, R. H. Carter, J. F. Kearney. 1997. Marginal zone B cells exhibit unique activation, proliferative and immunoglobulin secretory responses. Eur. J. Immunol. 27: 2366-2374. [Medline]
  27. Oliver, A. M., F. Martin, J. F. Kearney. 1999. IgMhighCD21high lymphocytes enriched in the splenic marginal zone generate effector cells more rapidly than the bulk of follicular B cells. J. Immunol. 162: 7198-7207. [Abstract/Free Full Text]
  28. Bourke, E., D. Bosisio, J. Golay, N. Polentarutti, A. Mantovani. 2003. The toll-like receptor repertoire of human B lymphocytes: inducible and selective expression of TLR9 and TLR10 in normal and transformed cells. Blood 102: 956-963. [Abstract/Free Full Text]
  29. Dasari, P., I. C. Nicholson, G. Hodge, G. W. Dandie, H. Zola. 2005. Expression of toll-like receptors on B lymphocytes. Cell. Immunol. 236: 140-145. [Medline]
  30. Mansson, A., M. Adner, U. Hockerfelt, L. O. Cardell. 2006. A distinct Toll-like receptor repertoire in human tonsillar B cells, directly activated by PamCSK, R-837, and CpG-2006 stimulation. Immunology 118: 539-548. [Medline]
  31. Genestier, L., M. Taillardet, P. Mondiere, H. Gheit, C. Bella, T. Defrance. 2007. TLR agonists selectively promote terminal plasma cell differentiation of B cell subsets specialized in thymus-independent responses. J. Immunol. 178: 7779-7786. [Abstract/Free Full Text]
  32. Cinamon, G., M. Matloubian, M. J. Lesneski, Y. Xu, C. Low, T. Lu, R. L. Proia, J. G. Cyster. 2004. Sphingosine 1-phosphate receptor 1 promotes B cell localization in the splenic marginal zone. Nat. Immunol. 5: 713-720. [Medline]
  33. Muller, U., U. Steinhoff, L. F. Reis, S. Hemmi, J. Pavlovic, R. M. Zinkernagel, M. Aguet. 1994. Functional role of type I and type II interferons in antiviral defense. Science 264: 1918-1921. [Abstract/Free Full Text]
  34. Adachi, O., T. Kawai, K. Takeda, M. Matsumoto, H. Tsutsui, M. Sakagami, K. Nakanishi, S. Akira. 1998. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9: 143-150. [Medline]
  35. Groeneveld, P. H., T. Erich, G. Kraal. 1985. In vivo effects of LPS on B lymphocyte subpopulations: migration of marginal zone-lymphocytes and IgD-blast formation in the mouse spleen. Immunobiology 170: 402-411. [Medline]
  36. Lu, T. T., J. G. Cyster. 2002. Integrin-mediated long-term B cell retention in the splenic marginal zone. Science 297: 409-412. [Abstract/Free Full Text]
  37. Gururajan, M., J. Jacob, B. Pulendran. 2007. Toll-like receptor expression and responsiveness of distinct murine splenic and mucosal B-cell subsets. PLoS ONE 2: e863
  38. Akira, S., K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4: 499-511. [Medline]
  39. Shiow, L. R., D. B. Rosen, N. Brdickova, Y. Xu, J. An, L. L. Lanier, J. G. Cyster, M. Matloubian. 2006. CD69 acts downstream of interferon-{alpha}β to inhibit S1P1 and lymphocyte egress from lymphoid organs. Nature 440: 540-544. [Medline]
  40. Bekeredjian-Ding, I., S. Inamura, T. Giese, H. Moll, S. Endres, A. Sing, U. Zahringer, G. Hartmann. 2007. Staphylococcus aureus protein A triggers T cell-independent B cell proliferation by sensitizing B cells for TLR2 ligands. J. Immunol. 178: 2803-2812. [Abstract/Free Full Text]
  41. Heer, A. K., A. Shamshiev, A. Donda, S. Uematsu, S. Akira, M. Kopf, B. J. Marsland. 2007. TLR signaling fine-tunes anti-influenza B cell responses without regulating effector T cell responses. J. Immunol. 178: 2182-2191. [Abstract/Free Full Text]
  42. Jegerlehner, A., P. Maurer, J. Bessa, H. J. Hinton, M. Kopf, M. F. Bachmann. 2007. TLR9 signaling in B cells determines class switch recombination to IgG2a. J. Immunol. 178: 2415-2420. [Abstract/Free Full Text]
  43. Jiang, W., M. M. Lederman, C. V. Harding, B. Rodriguez, R. J. Mohner, S. F. Sieg. 2007. TLR9 stimulation drives naive B cells to proliferate and to attain enhanced antigen presenting function. Eur. J. Immunol. 37: 2205-2213. [Medline]
  44. Johnston, D., B. Zaidi, J. C. Bystryn. 2007. TLR7 imidazoquinoline ligand 3M-019 is a potent adjuvant for pure protein prototype vaccines. Cancer Immunol. Immunother. 56: 1133-1141. [Medline]
  45. Chikumi, H., S. Fukuhara, J. S. Gutkind. 2002. Regulation of G protein-linked guanine nucleotide exchange factors for Rho, PDZ-{rho}GEF, and LARG by tyrosine phosphorylation: evidence of a role for focal adhesion kinase. J. Biol. Chem. 277: 12463-12473. [Abstract/Free Full Text]
  46. Chikumi, H., A. Barac, B. Behbahani, Y. Gao, H. Teramoto, Y. Zheng, J. S. Gutkind. 2004. Homo- and hetero-oligomerization of PDZ-{rho}GEF, LARG and p115{rho}GEF by their C-terminal region regulates their in vivo Rho GEF activity and transforming potential. Oncogene 23: 233-240. [Medline]
  47. Steinman, R. M., H. Hemmi. 2006. Dendritic cells: translating innate to adaptive immunity. Curr. Top. Microbiol. Immunol. 311: 17-58. [Medline]
  48. Pettersen, J. C., D. F. Borgen, K. C. Graupner. 1967. A morphological and histochemical study of the primary and secondary immune responses in the rat spleen. Am. J. Anat. 121: 305-317. [Medline]
  49. Gray, D., D. S. Kumararatne, J. Lortan, M. Khan, I. C. MacLennan. 1984. Relation of intra-splenic migration of marginal zone B cells to antigen localization on follicular dendritic cells. Immunology 52: 659-669. [Medline]
  50. Liu, L., B. R. Schwartz, N. Lin, R. K. Winn, J. M. Harlan. 2002. Requirement for RhoA kinase activation in leukocyte de-adhesion. J. Immunol. 169: 2330-2336. [Abstract/Free Full Text]



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