The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rodo, J.
Right arrow Articles by Penha-Gonçalves, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rodo, J.
Right arrow Articles by Penha-Gonçalves, C.
Right arrowPubmed/NCBI databases
*Substance via MeSH
The Journal of Immunology, 2006, 177: 4620-4626.
Copyright © 2006 by The American Association of Immunologists, Inc.

MHC Class II Molecules Control Murine B Cell Responsiveness to Lipopolysaccharide Stimulation1

Joana Rodo, Lígia A. Gonçalves, Jocelyne Demengeot, António Coutinho and Carlos Penha-Gonçalves2

Instituto Gulbenkian de Ciência, Oeiras, Portugal


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
LPS is a strong stimulator of the innate immune system and inducer of B lymphocyte activation. Two TLRs, TLR4 and RP105 (CD180), have been identified as mediators of LPS signaling in murine B cells, but little is known about genetic factors that are able to control LPS-induced cell activation. We performed a mouse genome-wide screen that aside from identifying a controlling locus mapping in the TLR4 region (logarithm of odds score, 2.77), also revealed that a locus closely linked to the MHC region (logarithm of odds score, 3.4) governed B cell responsiveness to LPS stimulation. Using purified B cells obtained from MHC congenic strains, we demonstrated that the MHCb haplotype is accountable for higher cell activation, cell proliferation, and IgM secretion, after LPS stimulation, when compared with the MHCd haplotype. Furthermore, B cells from MHC class II–/– mice displayed enhanced activation and proliferation in response to LPS. In addition, we showed that the MHC haplotype partially controls expression of RP105 (a LPS receptor molecule), following a pattern that resembles the LPS responsiveness phenotype. Together, our results strongly suggest that murine MHC class II molecules play a role in constraining the B cell response to LPS and that genetic variation at the MHC locus is an important component in controlling B cell responsiveness to LPS stimulation. This work raises the possibility that constraining of B cell responsiveness by MHC class II molecules may represent a functional interaction between adaptive and innate immune systems.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lipopolysaccharide is a fundamental structural component of the cell wall of Gram-negative bacteria (1). LPS harbors a conserved molecular pattern (lipid-A) that is recognized by innate immune system receptors that trigger the initial stages of antimicrobial host defense (2, 3). The course of infection mediated by Gram-negative bacteria is largely determined by the immune responses to LPS (4, 5). Although activation of the immune system during microbial invasion is generally protective, when excessive the reaction may harm the host through a maladaptive release of endogenously generated inflammatory mediators resulting in a life-threatening condition known as septic shock (6, 7).

The identification of genetic factors involved in the LPS response was initiated when allelic mutations of a single locus (LPS locus) in mice were shown to entirely abrogate the response to LPS (8, 9, 10). Later identification of the LPS gene through positional cloning (11) and production of TLR4 knockout mice confirmed that TLR4 (formerly known as the LPS locus) is needed to trigger the immune response to LPS (12, 13, 14). Those studies established that the signal-transducing receptor for LPS is TLR4. TLR4 belongs to the TLR family, a group of germline-encoded receptors that play a major role in innate immune recognition (15, 16, 17).

LPS recognition via TLR4 is complex and requires the help of different accessory molecules (15, 16, 17). After microbial infection, LPS associates with LPS-binding circulating proteins (18, 19). Although the exact mechanism of LPS recognition at the cell surface has not yet been elucidated, different studies propose that LPS associates with a TLR4/MD-2/CD14 complex (15, 16, 17, 20, 21, 22). After TLR4 activation by LPS, the MyD88 molecule is recruited to the plasma membrane, initiating a signaling cascade that leads to nuclear translocation of the transcription factors NF-{kappa}B and AP-1 (23) that in turn drive the production of TNF-{alpha} and cytokines such as IL-6. Engagement of TLR4 can also induce a MyD88-independent signaling transduction pathway, resulting in a delayed activation of NF-{kappa}B (16, 24, 25).

Recent studies have revealed that another TLR family receptor, RP105 (CD180), plays a role in B cell response to LPS (26, 27). In fact, the null mutation in the RP105 gene revealed impaired proliferative and humoral immune responses of RP105-deficient B cells to LPS (26). In parallel with TLR4, the surface expression and signaling of which seems to depend on the coexpression of the secreted extracellular protein MD-2, surface expression of RP105 is dependent on the coexpression of the MD-2 homolog MD-1 (21, 28). The RP105/MD-1 complex is preferentially expressed on the surface of mature B cells, but the mechanism through which RP105-MD-1 and TLR4 receptors cooperate in LPS response in B cells remains to be elucidated (26).

The dual ability of B cells to generate an Ag-specific immune response and to be activated through Toll-like receptors, establishes B cells as an important bridge between the innate and adaptive immune systems. In fact, LPS stimulation of murine B cells enhances the Ag presentation ability and is accompanied by B cell proliferation and secretion of large quantities of LPS-neutralizing Abs (29, 30). Although LPS has been used as a potent B cell stimulator, the molecular mechanisms involved in triggering B cell activation by LPS are poorly described.

Susceptibility to LPS in both humans and mice has been associated with allelic variations at the TLR4 locus (31). However, susceptibility to infections is usually under complex genetic control, and it is possible that naturally occurring allelic variation at different genes controls the response to LPS. With the intent to identify natural polymorphisms in genes that control the response to LPS, we performed a genome-wide search using the F2 intercross generation of the LPS high responder mouse strain C57BL/6 and the low responder mouse strain BALB/c. Our results revealed that the MHC class II locus is a major genetic factor in controlling B cell responsiveness to LPS.


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

The C57BL/6 and BALB/c mice were bred and maintained in conventional housing at the Instituto Gulbenkian de Ciência. All mice analyzed in this study were sex-matched adults. F1 mice were generated in reciprocal crosses between C57BL/6 and BALB/c and F1 (C57BL/6 x BALB/c) mice were used to generate an F2 progeny. B6.C-H2<d>/b By is a congenic strain carrying the MHCd haplotype in the C57BL/6 genetic background and is here referred to as B6.C. The C.B10-H2-<b>/LilMcd is a congenic strain carrying the MHCb haplotype in the BALB/c genetic background and is here referred to as C.B10. Both strains where purchased from The Jackson Laboratory. MHC class II–/– mice and MHC class II–/– transgenic (Tg) Ead (referred to as I-A–/– Tg Ead) mice, both on the C57BL/6 genetic background, were a gift of Dr. A. Bandeira (Institut Pasteur, Paris, France). The Tg Ead mice express the class II E molecule exclusively in the thymus.

All procedures were in accordance with national regulations on animal experimentation and welfare.

LPS and Abs

Commercial LPS from Salmonella typhimurium was purchased from Sigma-Aldrich. Purified LPS was extracted from commercial LPS using a phenol re-extraction method as described by Hirschfeld et al. (32). Purification of LPS eliminates the ability of signaling through murine TLR2 that is commonly observed in nonpurified LPS preparations (32). LPS purification was validated by the inability to induce cell response in splenocytes from TLR4null mice (data not shown). Anti-mouse CD19-FITC (1D3), anti-mouse CD45/B220-FITC (RA3-6B2), anti-mouse CD69-PE (H1.2F3), and streptavidin-PE were purchased from BD Pharmingen. Goat anti-mouse IgM UNLB Ab (1020-01) and goat anti-mouse IgM AP-coupled Ab (1020-40) were purchased from Southern Biotechnologies Associates. Anti-mouse CD180 (RP105) (RP/14) was purchased from eBioscience.

Cell purification

Single-cell suspensions from spleen and draining lymph nodes were prepared by straining of the tissues through a nylon mesh. Purified B cell populations were obtained by high speed sorting (MoFlo; Cytomation) after labeling using anti-mouse CD19 or B220 Abs. The purity of sorted cells was always >98%.

Cell activation

Single-cell suspensions from spleen, draining lymph nodes, and purified B cell populations were cultured at 37°C with 5% of CO2 for 24 h. Cells were plated at a density of 5 x 105 per well in 96-well flat-bottom plates with RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 10 mM HEPES (pH 7.4), 50 µM 2-ME, 100 U penicillin, and 100 µg/ml streptomycin (all from Invitrogen Life Technologies) in the presence or absence of purified or commercial LPS. Surface staining after activation used anti-mouse CD69-PE and anti-mouse CD19-FITC or CD45/B220-FITC Abs. Dead cells were excluded from analysis with propidium iodide. All data were acquired with a FACSCalibur cytometer (BD Biosciences) and analyzed with CellQuest (BD Biosciences) and FlowJo (Tree Star) software.

B cell proliferation assay

Purified B220+ cells (5 x 104) from spleen and draining lymph nodes were cultured in flat-bottom 96-well plates with or without 10 µg/ml purified LPS, as described above. After 42 h, cultures were pulsed with [3H]thymidine (ICN; sp. act., 5 Ci/mol), cells were harvested 6 h later, and thymidine incorporation was measured following conventional techniques. Data represent the mean and SD of six cultures expressed as cpm per culture.

ELISPOT

Numbers of IgM secreting cells were enumerated by a modification (32) of the Sedgwick and Holt technique (34). Briefly, 5 x 104 purified B220+ cells from spleen or draining lymph nodes were cultured in flat-bottom 96-well plates with or without 10 µg/ml purified LPS for 4 days. At the fifth day, one-half of the culture from each well was plated in serial dilutions in 96-well ELISA plates precoated with goat anti-mouse IgM unlabeled Ab (5 µg/ml). Plates were then incubated overnight at 37°C in 5°CO2. After three washes with PBS and PBS-Tween 20, goat anti-mouse IgM AP-coupled Ab was added, and plates were incubated overnight at 4°C. After three more washes with PBS-Tween 20, the aminomethyl propanol-5-bromo-4-chloro-3-indolyl phosphate substrate solution was added, and plates were again incubated overnight in the dark at 4°C. Plates were washed with distilled water, and blue spots were counted under a microscope and expressed as the number of IgM-secreting cells.

Genotyping

Genomic DNA was extracted from mouse tails according to standard procedures. A 142 progeny from the F2 (BALB/c x C57BL/6) generation was genotyped using conventional PCR protocols for 82 microsatellite markers, polymorphic for the parental strains. Markers were chosen according to their chromosomal position as given by the Whitehead MIT Centre for Genome Research (Cambridge, MA), to cover uniformly the whole of the mouse genome. The genotypes for each locus were determined by DNA marker amplification through conventional PCR techniques. Amplification products were analyzed in agarose gels (Cambrex BioScience Baltimore) with concentrations varying between 0.8 and 4%, according to the length of the polymorphism of the amplified fragment and stained with ethidium bromide (Sigma-Aldrich). For each marker, each individual of the F2 generation was typed as homozygous for one or the other of the parental strains or as heterozygous when presenting both the parental strain alleles.

Statistical analyses

Comparison of the means for the LPS-induced B cell activation phenotype among C57BL/6, BALB/c, and F1 mouse strains was performed using one-way ANOVA. Evidence of significant differences between each group (2 df) was considered for p ≤ 0.01. Quantitative trait locus analysis was performed using the R/QTL software (35). This program calculates logarithm of odds (LOD)3 scores over intervals between linked markers, representing the likelihood of genetic association for markers along the chromosome with the phenotype. The program also compares recessive, dominant, and additive models of allele actions. Levels of statistical significance were determined from permutation tests, and significant linkage (36) was considered for LOD ≥ 3.4 (p <0.001), whereas suggestive linkage was considered for LOD ≥ 1.9 (p <0.05).

Where applicable, the Student t test was used to evaluate statistically significant results. The following symbols were used to represent significance levels: *, p < 0.05; **, p < 0.01; ***, p < 0.001.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Genetic mapping of LPS-induced B cell activation

B cell response to LPS stimulation in vitro includes up-regulation of activation surface markers, cell proliferation, and IgM secretion. We observed that the proportion of CD69 expression among CD19+ splenic B cells after 24 h of LPS stimulation in vitro (Fig. 1) was consistently higher in the C57BL/6 mice (95.1 ± 1.7%) as compared with the BALB/c mice (68.9 ± 8.2%), whereas the F1 (C57BL/6 x BALB/c) generation presented an intermediate phenotype (87 ± 4.2%). To study the genetic control of LPS-induced B cell activation, we analyzed an F2 (C57BL/6 x BALB/c) progeny and found that the phenotype segregated in a continuous distribution that included low responders and high responders to the LPS stimulus. These data indicate that LPS-induced B cell activation was under polygenic control (Fig. 2), and we pursued a genome-wide search for genetic factors controlling the CD69 expression trait. Thus, the genotypes of 139 mice belonging to the F2 progeny were determined for 82 DNA markers that were chosen to cover the mouse genome with average density of 20 cM. Quantitative trait loci (QTL) analysis was performed using the R/QTL software and revealed that two chromosomal regions showed linkage to the LPS-induced activation phenotype measured by CD69 surface expression in CD19+ splenocytes (Fig. 3). One locus mapped to chromosome 4, and the highest associated marker was D4MIT301 (42.5 cM) with a LOD score of 2.77. This marker shows close linkage to the TLR4 locus (33.0 cM). It is likely that the TLR4 polymorphisms previously described between C57BL/6 and BALB/c mouse strains are associated with functional differences that could contribute to the observed LPS response phenotype. Unexpectedly, we found that the second controlling locus mapped to the MHC region on mouse chromosome 17, with the highest LOD score of 3.4 at marker D17MIT24 (20.4 cM; Fig. 3A). No evidence for linkage to any other chromosomal region was found. The C57BL/6-derived allele on the chromosome 4 locus shows an additive effect on the phenotype, whereas the C57BL/6-derived allele mapping to the MHC region on chromosome 17 exhibited recessive behavior (Fig. 3B). The C57BL/6 alleles at both loci increase the phenotype (Fig. 3B), strongly suggesting that they have a joint effect in LPS responsiveness phenotype of the C57BL/6 strain, but the nature of this interaction remains to be determined. It is known that commercial preparations of LPS can be contaminated with other bacterial products able to induce cell activation. Therefore, we extracted purified LPS to obtain a preparation that exclusively activated B cells through the LPS-binding receptors. We noted that the observed difference in response of C57BL/6 and BALB/c mouse strains was more pronounced when using purified LPS, implying that the phenotype under analysis was linked to B cell activation through LPS-binding receptors (Fig. 4).


Figure 1
View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 1. The LPS-induced activation phenotype. Flow cytometry profiles of CD69 expression in CD19+ splenocytes representative of C57BL/6, BALB/c and F1 (C57BL/6 x BALB/c) mice after stimulation in vitro with 10 µg/ml commercial LPS for 24 h. The values in the upper right quadrant indicate the percentage of the CD19+ population that was considered to be activated as compared with nonstimulated controls.

 

Figure 2
View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 2. Segregation of the LPS-induced activation phenotype. Percentage of CD69 expressing splenocytes was analyzed as in Fig. 1. Data refer to 8 C57BL//6 ({blacksquare}), 10 BALB/c ({triangleup}), 18 F1 (9 BALB/c x C57BL/6 and 9 C57BL/6 x BALB/c) ({blacktriangledown}) and 139 F2 (BALB/c x C57BL/6; {square}) mice.

 

Figure 3
View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 3. QTL analysis of the LPS-induced activation phenotype. A, LOD score profiles for the two chromosomes that showed significant linkage to the CD69 expression trait. Genetic maps based on data from 139 F2 mice of chromosomes 4 and 17 were generated by R/QTL software, and LOD profiles represent the maximum likelihood estimates for the presence of a QTL at each point of the analyzed regions. B, Genetic effect plots of marker D4Mit301 on chromosome 4 and D17Mit24 on chromosome 17 on the LPS-induced CD69 expression phenotype. Each data point represents the average trait value for the group of F2 (BALB/c x B57BL/6) mice with the indicated genotype (CC homozygous for BALB/c, CB heterozygous, and BB homozygous for C57BL/6). Bars, SE.

 

Figure 4
View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 4. Receptor triggering by purified LPS reproduces LPS-induced activation phenotype. Analysis of CD69 expression in B220+ cells after 24 h stimulation with 10 µg/ml commercial and purified LPS preparations. Data refer to the average values of spleen cells from four C57BL/6 and four BALB/c mice. LPS was purified and tested for TLR4 requirement as described in Materials and Methods.

 
Congenic mapping confirms a LPS-responsive region in close linkage with the MHC locus

To confirm that the locus identified in close linkage to the MHC genetic region had a role in LPS-induced B cell response, we analyzed different responsiveness traits in purified B220+ splenic cells derived from two MHC-congenic mouse strains, B6.C and C.B10 (Fig. 5). The results showed that introgression of a fragment of 11.1 cM of chromosome 17, comprising the MHC genetic region from the BALB/c strain onto a C57BL/6 genetic background (B6.C-congenic strain; Fig. 5) confers decreased expression of CD69 (Fig. 6A), cell proliferation (Fig. 6C), and IgM secretion (Fig. 6E) in LPS-stimulated B cells as compared with progenitor C57BL/6 cells. Albeit at lower responsiveness level, the introgression of an analogous fragment of 7.4 cM from the C57BL/10 strain onto a BALB/c genetic background (C.B10-congenic strain; Fig. 5) confers higher frequency of CD69-expressing cells (Fig. 6B), increased cell proliferation (Fig. 6D), and higher numbers of IgM-secreting cells (Fig. 6F) after LPS stimulation, as compared with parental BALB/c mice. The low LPS responsiveness to purified LPS observed in both BALB/c and C.B10 strains (Fig. 6, B, D, and F) is probably due to the sharing of the TLR4 allele from BALB/c which is not represented in the C57BL/6 and B6.C strains (Fig. 6, A, C, and E). Thus, these data suggest that even in the BALB/c low LPS-responsive genetic background, the MHC haplotype has a detectable effect on the responsiveness to LPS (Fig. 6. B, D, and F). The observed differences in congenic strains are not attributable to differences in the surface expression level of MHC class II molecules (data not shown), suggesting that MHC molecules expression level is not involved in the LPS responsiveness phenotype. These results confirm that allelic variants of the region on chromosome 17 spanning the MHC locus contain genetic factor(s) that partially restore(s) the difference in CD69 expression observed in LPS-stimulated B cells of the parental mouse strains. Furthermore, the results show that the action of such a genetic factor, other than controlling LPS-induced B cell activation, exhibits a measurable effect at the level of B cell proliferation and Ab production, suggesting that genetic variation in this region may influence the course of the LPS immune response at the systemic level.


Figure 5
View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 5. Congenic chromosome maps for B6.C and C.B10 mouse strains. The C.B10-congenic region encompasses the MHC genetic region derived from the C57BL/10 mouse strain in a BALB/c genetic background. The B6.C-congenic region encompasses the MHC genetic region derived from the BALB/c mouse strain in a C57BL/6 genetic background. Bars, chromosome 17 of the C57BL/6, BALB/c, B6.C, and C.B10 mouse strains. White and black areas are regions that are derived from the BALB/c and C57BL/6 strains, respectively; Pos, chromosomal position.

 

Figure 6
View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 6. Congenic mapping of a LPS responder region that includes the MHC locus. The LPS response was measured in B220+-purified spleen cells as percentage of CD69 expressing B220+ cells (A and B), thymidine incorporation after 42 h of incubation (C and D), and number of IgM-secreting cells measured by ELISPOT (E and F). The cells were activated in the presence or absence of purified LPS (10 µg/ml). Data refer to the average values of pooled splenocytes from five C57BL/6, five B6.C, 5 C.B10, and five BALB/c mice. Values are representative of three experiments performed in separate occasions, and statistically significant results are marked by stars as described in Materials and Methods.

 
Expression of MHC class II molecules limits B cell response to LPS

MHC class II genes are included in the congenic region of the C.B10- and B6.C-congenic strains (Fig. 5) and have been claimed to participate in the response to LPS (37). To test whether MHC class II genes are implicated in LPS-induced activation of B cells, we compared the response of B220+-purified cells from spleen and lymph nodes, in MHC class II–/– and C57BL/6 mice. Purified B cells from MHC class II–/– mice showed a higher response to LPS than C57BL/6 mice, translated in higher percentage of CD69+ B cells, higher cell proliferation and higher numbers of IgM-secreting cells (Fig. 7). These results indicate that the lack of expression of MHC class II genes in B cells leads to increased B cell response to LPS receptor triggering. To show that higher response to LPS in MHC class II–/– mice was not due to absence of CD4 T cells, we tested I-A–/– Tg Ead mice which express MHC class II molecules exclusively in the thymus and show restoration of the CD4 T cell compartment. In fact, purified B220+ cells from such mice do not restore wild-type response to LPS, strongly suggesting that the higher LPS response of MHC class II–/– mice is due to the lack of MHC class II molecules on B cells (Fig. 8).


Figure 7
View larger version (10K):
[in this window]
[in a new window]
  		FIGURE 7. B cells of MHC class II–/– mice show enhanced response to LPS. Spleen as well as draining lymph node B220+ cells were tested for the LPS response as described in Fig. 6. A, CD69 expression; B, thymidine incorporation; C, number of IgM-secreting cells. Data refer to the average values of pooled spleen and draining lymph node cells from five C57BL/6 and five MHC II–/– mice. Values are representative of four experiments performed on separate occasions, and statistically significant results are marked by stars as described in Materials and Methods.

 

Figure 8
View larger version (7K):
[in this window]
[in a new window]
  		FIGURE 8. LPS response of MHC class II–/– B cells generated in presence of a restored CD4 T cell compartment. The LPS response of purified B220+ splenocytes was measured as the percent of CD69-expressing cells after 24 h of incubation in the presence or absence of purified LPS (5 µg/ml). Data refer to the average values of pooled splenocytes from five C57BL/6, five MHC class II–/–, and five MHC class II–/– Tg Ea mice, which express the MHC class II E molecule exclusively in the thymus and therefore carry a restored CD4 T cell compartment. Values are representative of two independent experiments, and statistically significant results are marked by stars as described in Materials and Methods.

 
The MHC haplotype controls expression of RP105 (CD180)

In search for molecular components that could explain the observed MHC control of B cell responsiveness to LPS stimulation, we studied the surface expression of RP105 on freshly isolated B cells. RP105 is a LPS-responsive TLR molecule that is mainly expressed in mature B cells. Our results revealed that expression of RP105 measured both as percent of positive cells and mean fluorescence intensity correlates with MHC haplotype (Fig. 9). In fact, B6.C mice show lower levels of RP105 expression than C57BL/6 mice, whereas C.B10 mice show higher levels than BALB/c mice. Such results suggest that along with unidentified genetic background factors, the MHC haplotype has on its own a measurable effect on the surface expression of RP105. In accordance with the observed LPS responsiveness phenotype, the MHC class II–/– mouse B cells also showed an up-regulation of RP105 expression (data not shown), reinforcing the notion of a functional link between MHC class II gene products and B cell responsiveness to LPS. We also measured TLR4 RNA expression in nonstimulated purified B cells, but the observed differences between MHC congenic strains and their parental strains did not reach statistical significance (data not shown). However, it remains to be determined whether TLR4 surface expression in these mouse strains follows the observed RP105 RNA expression patterns.


Figure 9
View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 9. The MHC haplotype contributes to control RP105 (CD180) expression on B cells. Expression of RP105 was measured in freshly isolated splenocytes in five C57BL/6, four B6.C, four C.B10, and five BALB/c mice. A, Representative flow cytometry profiles of the percent of RP105+ cells among B220+ population in each mouse strain. B, Average values and SDs for RP105 mean fluorescence intensity (M.F.I.) among B220+ cells for each mouse strain. Values are representative of two independent experiments, and statistically significant results are marked by stars as described in Materials and Methods.

 
The results here reported show that MHC class II molecules are involved in B cell activation via LPS-binding receptors and indicate that polymorphisms at the MHC class II genes constrain B cell responsiveness to LPS stimulation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The molecular mechanisms leading to innate stimuli recognition, signaling transduction and subsequent cellular events in B cells, have been extensively investigated. However, there is little information on genetic components mediating the cross-talk of cellular innate immune functions and adaptive immune functions in B cells. This genetic study shows that the difference in LPS responsiveness observed between the C57BL/6 and BALB/c mouse strains was linked to two genetic regions, the TLR4 region and the MHC locus. Although it is expected that the TLR4 region would be linked to this phenotype, it was surprising to verify that the MHC locus showed the strongest linkage (LOD score, 3.4) and had a genetic effect comparable with that of the TLR4 region in controlling LPS responsiveness (Fig. 3). In fact, we show that genetic variation of the MHC haplotype as well as lack of expression of MHC class II molecules control the efficiency of B cell response to LPS stimulation as measured by activation marker expression, cell proliferation, and IgM secretion.

The notion that MHC class II somehow interferes with B cell response to LPS has been reported earlier (30), and a recent study proposes that the LPS responsiveness in other cell types such as human PBMC and mouse macrophages is also affected by the absence of MHC class II expression (37). Interestingly, expression of MHC class II molecules was reported to enable LPS response of those cell types, whereas we observed a decreased B cell response to LPS. This discrepancy suggests that the mechanism through which MHC class II molecules interfere with the LPS response may be different in different cell types.

Our results have demonstrated that MHC class II genetic configurations displaying lower responsiveness to LPS also show lower surface expression of the LPS recognition complex component RP105 (CD180). This finding favors the hypothesis that action of MHC class II molecules in constraining B cell ability to respond to LPS is modulated through surface expression of RP105, in such a way that better surface expression of RP105 leads to better responsiveness to LPS. The mechanism by which such modulation occurs remains to be clarified. One possibility is that RP105 expression at the cell surface is down-regulated in B cells that have experienced Ag presentation through the MHC class II pathway. Such a possibility would support a model where Ag presentation and T cell interaction would render B cells short or long term committed to adaptive functions and poor responders to LPS stimulation. The finding that MHC class II genetic configuration plays a role in RP105 controlling cell surface suggests that they may be as well involved in the controlling expression of other components of the innate immune system. However, quantification of TLR4 RNA in unstimulated B cells was not informative to ascertain whether polymorphisms in the MHC II genes constrain B cell responsiveness to LPS via TLR4 surface expression.

Although the data presented here suggest that MHC controlled LPS responsiveness is at least in part explained by modulation of innate immunity molecules expression, it is not excluded that other mechanisms could also exist that explain additional roles of MHC class II molecules in B cell response to LPS. In particular, our results do not exclude the hypothesis that the MHC II pathway could also have inhibitory effects on LPS responsiveness at the level of surface recognition or intracellular signaling. Different lines of evidence suggest that recognition of LPS at cell surface may be affected by interactions with MHC class II molecules. Some early studies have shown that monoclonal anti I-A/E Abs directly inhibit B cell responses to LPS with a gradual loss of inhibitory activity over time (30, 38, 39). It is possible that these Abs may mediate their effect either by blocking the LPS receptor complex at the cell membrane or because binding to Ia Ags would result in the redistribution of other surface molecules, including LPS receptors (30). Our results would also support the hypothesis that MHC II pathway could have inhibitory effects on the LPS pathway at the level of intracellular signaling. On the other hand, MHC class II molecules could directly interact with LPS, leading to decreased cell activation. Several studies have shown that some bacterial polysaccharides are capable of binding MHC class II molecules inside APCs and are presented to T cells through the MHC class II endocytic pathway (40). The capacity of polysaccharides to induce T cell responses opens new perspectives on the MHC class II presentation paradigm. Although there is no evidence that LPS or LPS-derived molecules are presented to T cells through the MHC class II complex, Forestier et al. (41) demonstrated that different types of internalized LPS accumulate in an MHC class II-positive lysosomal compartment in both murine and human B cells. The same study shows that Brucella abortus LPS coprecipitates with MHC class II molecules in an haplotype-independent manner, constituting evidence for a direct interaction between the MHC class II molecules and LPS.

The action of MHC class II molecules in B cell activation by LPS is still not clear. Different studies have shown an existing relationship between LPS and the MHC class II gene products at different levels but have not come together to elucidate the nature of such interaction. We provided genetic evidence confirming that MHC class II molecules constrain B cell response to LPS and that this occurs in an allelic-dependent manner. These findings may have implications to the study of human susceptibility to innate stimuli by pathogens.


   Acknowledgments
 
We thank Dr. António Bandeira for making available to us the MHC class II Abnull mice and Tg MHC class II Ead mice. We thank Paulo Almeida for help with the statistical analyses. Margarida Almeida Santos and Dr. Íris Caramalho are acknowledged for advice on the experimental work. We thank Alexis González for his expertise in cell sorting.


   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 Fundação para a Ciência e Tecnologia Grant SAU-MMO/60964/2004. Back

2 Address correspondence and reprint requests to Carlos Penha-Gonçalves, Instituto Gulbenkian de Ciência, Apartado 14 P-2781-901, Oeiras, Portugal. E-mail address: cpenha{at}igc.gulbenkian.pt Back

3 Abbreviations used in this paper: LOD, logarithm of odds; Tg, transgenic; QTL, quantitative trait loci. Back

Received for publication February 23, 2006. Accepted for publication July 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Rietschel, E. T., H. Brade, O. Holst, L. Brade, S. Muller-Loennies, U. Mamat, U. Zahringer, F. Beckmann, U. Seydel, K. Brandenburg, A. J. et al. 1996. Bacterial endotoxin: chemical constitution, biological recognition, host response, and immunological detoxification. Curr. Top Microbiol. Immunol. 216: 39–81.
  2. Netea, M. G., M. van Deuren, B. J. Kullberg, J. M. Cavaillon, J. W. Van der Meer. 2002. Does the shape of lipid A determine the interaction of LPS with Toll-like receptors?. Trends Immunol. 23: 135-139. [Medline]
  3. Rietschel, E. T., H. W. Wollenweber, U. Zahringer, O. Luderitz. 1982. Lipid A, the lipid component of bacterial lipopolysaccharides: relation of chemical structure to biological activity. Klin. Wochenschr. 60: 705-709. [Medline]
  4. Shahin, R. D., I. Engberg, L. Hagberg, C. Svanborg Eden. 1987. Neutrophil recruitment and bacterial clearance correlated with LPS responsiveness in local Gram-negative infection. J. Immunol. 138: 3475-3480. [Abstract]
  5. Ulevitch, R. J., P. S. Tobias. 1999. Recognition of Gram-negative bacteria and endotoxin by the innate immune system. Curr. Opin. Immunol. 11: 19-22. [Medline]
  6. Rietschel, E. T., U. Schade, M. Jensen, H. W. Wollenweber, O. Luderitz, S. G. Greisman. 1982. Bacterial endotoxins: chemical structure, biological activity and role in septicaemia. Scand J. Infect. Dis. Suppl. 31: 8-21. [Medline]
  7. Abhishek, A., A. Anushree, T. Ashutosh, S. Ray. 2005. Comment to: Septic shock: current pathogenic concept from a clinical perspective Tsiotou AG, Sakorafas GH, Anagnostopoulos G, Bramis J Med Sci Monit, 2005; 11(3): RA76-RA85: future treatment perspectives in septic shock. Med. Sci. Monit. 11: LE22-LE23. [Medline]
  8. Coutinho, A., G. Moller, E. Gronowicz. 1975. Genetical control of B-cell responses. IV. Inheritance of the unresponsiveness to lipopolysaccharides. J. Exp. Med. 142: 253-258. [Abstract/Free Full Text]
  9. Poltorak, A., X. He, I. Smirnova, M. Y. Liu, C. Van Huffel, X. Du, D. Birdwell, E. Alejos, M. Silva, C. Galanos, et al 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 2085-2088. [Abstract/Free Full Text]
  10. Coutinho, A., L. Forni, F. Melchers, T. Watanabe. 1977. Genetic defect in responsiveness to the B cell mitogen lipopolysaccharide. Eur. J. Immunol. 7: 325-328. [Medline]
  11. Poltorak, A., I. Smirnova, X. He, M. Y. Liu, C. Van Huffel, O. McNally, D. Birdwell, E. Alejos, M. Silva, X. Du, et al 1998. Genetic and physical mapping of the Lps locus: identification of the toll-4 receptor as a candidate gene in the critical region. Blood Cells Mol. Dis. 24: 340-355. [Medline]
  12. Hoshino, K., O. Takeuchi, T. Kawai, H. Sanjo, T. Ogawa, Y. Takeda, K. Takeda, S. Akira. 1999. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162: 3749-3752. [Abstract/Free Full Text]
  13. Kalis, C., B. Kanzler, A. Lembo, A. Poltorak, C. Galanos, M. A. Freudenberg. 2003. Toll-like receptor 4 expression levels determine the degree of LPS-susceptibility in mice. Eur. J. Immunol. 33: 798-805. [Medline]
  14. Watson, J., K. Kelly, M. Largen, B.A. Taylor. 1978. The genetic mapping of defective LPS response gene in C3H/HeJ mice. J. Immunol. 120: 422-424. [Abstract/Free Full Text]
  15. Janeway, C. A., Jr, R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20: 197-216. [Medline]
  16. Akira, S., K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4: 499-511. [Medline]
  17. Medzhitov, R.. 2001. Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1: 135-145. [Medline]
  18. Wright, S. D., P. S. Tobias, R. J. Ulevitch, R. A. Ramos. 1989. Lipopolysaccharide (LPS) binding protein opsonizes LPS-bearing particles for recognition by a novel receptor on macrophages. J. Exp. Med. 170: 1231-1241. [Abstract/Free Full Text]
  19. Schumann, R. R., S. R. Leong, G. W. Flaggs, P. W. Gray, S. D. Wright, J. C. Mathison, P. S. Tobias, R. J. Ulevitch. 1990. Structure and function of lipopolysaccharide binding protein. Science 249: 1429-1431. [Abstract/Free Full Text]
  20. da Silva Correiaqq, J., K. Soldau, U. Christen, P. S. Tobias, R. J. Ulevitch. 2001. Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex transfer from CD14 to TLR4 and MD-2. J. Biol. Chem. 276: 21129-21135. [Abstract/Free Full Text]
  21. Shimazu, R., S. Akashi, H. Ogata, Y. Nagai, K. Fukudome, K. Miyake, M. Kimoto. 1999. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J. Exp. Med. 189: 1777-1782. [Abstract/Free Full Text]
  22. Akashi, S., R. Shimazu, H. Ogata, Y. Nagai, K. Takeda, M. Kimoto, K. Miyake. 2000. Cutting edge: cell surface expression and lipopolysaccharide signaling via the Toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages. J. Immunol. 164: 3471-3475. [Abstract/Free Full Text]
  23. Ahmad-Nejad, P., H. Hacker, M. Rutz, S. Bauer, R. M. Vabulas, H. Wagner. 2002. Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur. J. Immunol. 32: 1958-1968. [Medline]
  24. Kawai, T., O. Takeuchi, T. Fujita, J. Inoue, P. F. Muhlradt, S. Sato, K. Hoshino, S. Akira. 2001. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J. Immunol. 167: 5887-5894. [Abstract/Free Full Text]
  25. Toshchakov, V., B. W. Jones, P. Y. Perera, K. Thomas, M. J. Cody, S. Zhang, B. R. Williams, J. Major, T. A. Hamilton, M. J. Fenton, S. N. Vogel. 2002. TLR4, but not TLR2, mediates IFN-beta-induced STAT1{alpha}/beta-dependent gene expression in macrophages. Nat. Immunol. 3: 392-398. [Medline]
  26. Ogata, H., I. Su, K. Miyake, Y. Nagai, S. Akashi, I. Mecklenbrauker, K. Rajewsky, M. Kimoto, A. Tarakhovsky. 2000. The Toll-like receptor protein RP105 regulates lipopolysaccharide signaling in B cells. J. Exp. Med. 192: 23-29. [Abstract/Free Full Text]
  27. Miyake, K., Y. Yamashita, M. Ogata, T. Sudo, M. Kimoto. 1995. RP105, a novel B cell surface molecule implicated in B cell activation, is a member of the leucine-rich repeat protein family. J. Immunol. 154: 3333-3340. [Abstract]
  28. Miyake, K., R. Shimazu, J. Kondo, T. Niki, S. Akashi, H. Ogata, Y. Yamashita, Y. Miura, M. Kimoto. 1998. Mouse MD-1, a molecule that is physically associated with RP105 and positively regulates its expression. J. Immunol. 161: 1348-1353. [Abstract/Free Full Text]
  29. Kearney, J. F., A. R. Lawton. 1975. B lymphocyte differentiation induced by lipopolysaccharide. I. Generation of cells synthesizing four major immunoglobulin classes. J. Immunol. 115: 671-676. [Abstract/Free Full Text]
  30. Forsgren, S., G. Pobor, A. Coutinho, M. Pierres. 1984. The role of I-A/E molecules in B lymphocyte activation. I. Inhibition of lipopolysaccharide-induced responses by monoclonal antibodies. J. Immunol. 133: 2104-2110. [Abstract]
  31. Wells, C. A., T. Ravasi, G. J. Faulkner, P. Carninci, Y. Okazaki, Y. Hayashizaki, M. Sweet, B. J. Wainwright, D. A. Hume. 2003. Genetic control of the innate immune response. BMC Immunol. 4: 5[Medline]
  32. Hirschfeld, M., Y. Ma, J. H. Weis, S. N. Vogel, J. J. Weis. 2000. Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine Toll-like receptor 2. J. Immunol. 165: 618-622. [Abstract/Free Full Text]
  33. Grandien, A., A. Coutinho, J. Andersson. 1990. Selective peripheral expansion and activation of B cells expressing endogenous immunoglobulin in Mu-transgenic mice. Eur. J. Immunol. 20: 991-998. [Medline]
  34. Sedgwick, J. D., P. G. Holt. 1983. A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. J. Immunol. Methods 57: 301-309. [Medline]
  35. Broman, K. W., H. Wu, S. Sen, G. A. Churchill. 2003. R/qtl: QTL mapping in experimental crosses. Bioinformatics 19: 889-890. [Abstract/Free Full Text]
  36. Lander, E., L. Kruglyak. 1995. Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat. Genet. 11: 241-247. [Medline]
  37. Piani, A., J. P. Hossle, T. Birchler, C. A. Siegrist, D. Heumann, G. Davies, S. Loeliger, R. Seger, R. P. Lauener. 2000. Expression of MHC class II molecules contributes to lipopolysaccharide responsiveness. Eur. J. Immunol. 30: 3140-3146. [Medline]
  38. Forsgren, S., A. C. Martinez, A. Coutinho. 1987. The role of I-A/E molecules in B-lymphocyte activation. II. Mechanism of inhibition of the responses to lipopolysaccharide by anti-I-A/E antibodies. Scand. J. Immunol. 25: 225-234. [Medline]
  39. Santamaria, P., R. C. Gehrz, M. K. Bryan, J. J. Barbosa. 1989. Involvement of class II MHC molecules in the LPS-induction of IL-1/TNF secretions by human monocytes: quantitative differences at the polymorphic level. J. Immunol. 143: 913-922. [Abstract]
  40. Cobb, B. A., Q. Wang, A. O. Tzianabos, D. L. Kasper. 2004. Polysaccharide processing and presentation by the MHCII pathway. Cell 117: 677-687. [Medline]
  41. Forestier, C., E. Moreno, S. Meresse, A. Phalipon, D. Olive, P. Sansonetti, J. P. Gorvel. 1999. Interaction of Brucella abortus lipopolysaccharide with major histocompatibility complex class II molecules in B lymphocytes. Infect. Immun. 67: 4048-4054. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
K. Hannestad and H. Scott
The MHC Haplotype H2b Converts Two Pure Nonlupus Mouse Strains to Producers of Antinuclear Antibodies
J. Immunol., September 1, 2009; 183(5): 3542 - 3550.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rodo, J.
Right arrow Articles by Penha-Gonçalves, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rodo, J.
Right arrow Articles by Penha-Gonçalves, C.
Right arrowPubmed/NCBI databases
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS