G-protein-coupled receptors (GPCR) regulate signal transduction in cells, participating in a variety of biological processes . An activated GPCR makes a complex with a G-protein defined by α subunit. Gα subunits consist with Gαs, Gαi/o, Gαq and Gα12 . The members of Gα12 family consisting of Gα12 and Gα13 are critical mediators in regulating effectors or cellular responses. Their interactions with specific Rho guanine nucleotide exchanging factor result in small GTPase RhoA activation, leading to diverse biological functions such as migration and maturation of B cells, morphology and motility of cells, and smooth muscle contraction [2 - 4].
Activated B cells proliferate and differentiate into immunoglobulin (Ig)-producing plasma cells or long-lived memory cells . It is well recognized that Ig antibody (Ab) production by B cells is the critical process in the defense against infection. Immune cells express a variety of GPCRs, whose activations result in coupling of G-proteins for signal amplifications . In particular, it has been shown that Gα12 and Gα13 regulate the homeostasis of marginal zone B cells, in which the G-proteins activated LSC, a p115RhoGEF, for humoral responses . Our studies showed that activation of Gα12and/or Gα13 leads to the induction of iNOS and COX-2 through NF-κB, an essential transcription factor required for immune responses [6,7]. Moreover, sphingosine 1-phospate (S1P), a representative lysophospholipid GPCR ligand in blood or lymph nodes, disseminates the signal to B cells via S1P3 GPCR to position marginal zone B cells .
Despite the identified role of Gα12/Gα13 in the regulation of mariginal zone B cell biology , the functions of Gα12/Gα13 in Ig production have not been completely identified. In view of complex network of the immune system and involvements of diverse cell types, we investigated whether the Gα12 family members regulate the production of Ig classes and IgG subclasses with particular emphasis on their roles in thymus-dependent or thymus-independent immunity. In this study, Ab production was measured after challenges of two different types of antigens. To elicit thymus-dependent immune response, wild type (WT) or Gα12 and/or Gα13 heterozygous or homozygous knockout (KO) mice were immunized with ovalbumin (OVA). In another set, the animals were injected with trinitrophenyl-lipopolysaccharide (TNP-LPS) to promote thymus-independent Ig production. Through these in vivo analyses, it was found that Gα12 and Gα13 have regulatory functions in producing IgG subclasses. This information may bring insight in understanding the role of Gα12 members in humoral immune responses.
To determine the possible role(s) of Gα12 and/or Gα13 in the regulation of thymus-dependent Ab production, the Ig titers were measured in WT mice or mice deficient in Gα12/Gα13 that had been immunized with OVA and booster-injected with the same antigen 2 weeks after, which was followed by OVA nebulizations (Fig. 1A). After the immunizations and nebulizations, WT mice showed normal OVA-specific IgG and IgM production (Fig. 1A, Table 1). In contrast, homozygous absence of the Gα12 gene significantly impaired OVA-specific IgG production. When OVA-specific Ig subclass contents were assessed, Gα12 deficiency decreased the production of IgG1, IgG2a and IgG2b subclasses compared to those in WT. Also, double heterozygous deletions of Gα12 and Gα13 significantly reduced OVA-specific IgG content with decreases in the levels of IgG1, IgG2a or IgG2b subclasses. The increased production of IgM was unaffected by the absence of Gα12 and/or Gα13. In this assay, we could not use the animals with homozygous Gα13 KO because these mice die before birth . Our data demonstrated that Gα12 and Gα13 are both required for thymus-dependent IgG1, IgG2a and IgG2b production.
|Thymus-dependent Ig production||Thymus-independent Ig production|
|WT||Gα12 +/-||Gα12 -/-||Gα13 +/-||Gα12/Gα13+/-||WT||Gα12 +/-||Gα12 -/-||Gα13 +/-||Gα12/Gα13 +/-|
LPS is a classic mitogenic stimulus that activates the innate B cell receptors . It is known that LPS stimulation activates B cells without T cell help. In another set of experiments, we determined the roles of Gα12/Gα13 for the regulation of B cell immune response stimulated by TNP-LPS, a well-known thymus-independent antigen. In WT mice, total Ig production by TNP-LPS was greatly promoted 2 weeks after (Fig. 1B). The heterozygous or homozygous KO of Gα12 inhibited TNP-LPS-specific IgG production in a gene dose-dependent manner. In the mice partially deficient in Gα13 or both Gα12 and Gα13, the degrees of TNP-LPS-specific IgG production were also decreased compared to WT mice challenged with the same antigen (Fig. 1B, Table 1). We found that decreases in TNP-LPS-specific IgG titer by the lack(s) of Gα12 and/or both Gα12 and Gα13 exactly matched with decreases in TNP-LPS-specific IgG2b, indicating the specific role of Gα12 and Gα13 for the production of IgG2b subclass. TNP-LPS-specific IgG1, IgG2a, and IgM contents were not changed by the gene KOs, suggesting that the change in TNP-LPS-specific IgG production might be due to that in IgG2b. Thus, Ig production in response to TNP-LPS was found to be regulated by Gα12 and Gα13. All of these results provide evidence that Gα12 and Gα13 are both required for regulating thymus-dependent and thymus-independent production of IgG subclasses.
There are two distinctive pathways that induce B cell responses (that is, thymus-dependent and thymus-independent B cell activation) [10,11]. In the present study, we demonstrated for the first time that Gα12 and Gα13 are both required for thymus-dependent production of IgG subclasses. Significant changes in the humoral response, particularly in regulating IgG, by the lack(s) of Gα12 and Gα13 highlight the need to consider the G-protein pathway as one of important regulatory controls for T-cell dependent immune network.
Affinity maturation of B cells, which need Th1 cytokines, consists with the processes of clonal proliferation, somatic hypermutation, and selection . Th1 cytokines such as interferon-γ (IFN-γ) stimulate IgG2a, IgG2b, and IgG3 production . Therefore, our results showing that Gα12 and Gα13 deficiency notably decreased IgG2a and IgG2b subclasses in an OVA-immunized model suggest that the affinity maturation processes of B cells might be affected by Gα12 and Gα13. Th2 cytokine (e.g., interleukin-4) particularly induces Ig switching to IgG1 and IgE . Our data indicated that the titer of anti-OVA IgG1, but not anti-OVA IgE, was lowered by Gα12/Gα13 deficiency, which suggested that maturation of B cell to IgG1-producing plasma cell might need the G-protein-mediated signaling processes presumably upon stimulation of Th2 cytokine(s). Collectively, the essential regulatory function of Gα12/Gα13 in producing IgG subclasses lends support to the hypothesis that helper T cell-dependent B cell activation by antigen requires the G-protein-mediated signaling pathway.
Another type of B cell activation is thymus-independent, which is triggered by viral particles, common bacterial antigens, and TLR ligands without T cell help . B cell activation by thymus-independent antigens (e.g., LPS) causes the production of polyclonal antibody . In our animal model, TNP-LPS-inducible production of IgG2b, but not IgG3, was decreased by the absence of Gα12 and Gα13, showing that the G-proteins might regulate thymus-independent IgG production by B cells. Because transforming growth factor-β stimulates IgG2b and IgA production , the decrease in IgG2b in TNP-LPS-injected mice deficient in Gα12/Gα13 might be associated with repression of transforming growth factor-β. This possibility is strengthened by our finding that Gα12/Gα13 regulate transforming growth factor-β production in the liver of mice challenged with dimethylnitrosamine (Lee et al, unpublished data).
Gα12 regulates NF-κB-mediated COX-2 induction by S1P in a process mediated by the JNK-dependent ubiquitination and degradation of IκBα . Because antigen-induced cell signaling requires NF-κB and AP-1, decreased activation of the transcription factors may account for altered IgG production in the KO mice. Our results illustrating the role of Gα12 and Gα13 in IgG production may be explained by other possibilities: that is, (1) the role of Gα12/Gα13 in B cell reentry into the secondary lymphoid organ and (2) the regulatory role of Gα12/Gα13 in the specific step of Ab production.
OVA or TNP-LPS Immunizations
All experiments were conducted under the guidelines of the Institutional Animal Use and Care Committee at Seoul National University, Korea. WT and Gα12/Gα13 KO mice at the age of 8–10 weeks (25~30 g)(Gα12+/-, Gα12-/-, Gα13+/-, and Gα12/Gα13+/-) were used for in vivo experiments. To induce OVA-specific Ab production, the mixture of 100 μg endotoxin-free OVA (MP Biomedicals, Aurora, OH) dissolved in PBS and alum (Pierce, IL) was i.p. injected to the mice on day 1. On day 14, the mice were i.p. injected again with 100 μg OVA. The mice were subsequently exposed to aerosol of 1% OVA solution for 30 min once a day on days 26, 27 and 28. In another set of experiments, a single dose of TNP-LPS (Biosearch Technologies, Novato, CA) was i.p. injected to WT and Gα12/Gα13 KO mice to assess the production of TNP-LPS-specific Ab. The animals were bled 2 weeks after.
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA assays were performed to determine antigen-specific total IgG, IgG1, IgG2a, IgG2b, IgG3, IgA, IgE and IgM in aliquots of diluted serum (1:2000) in a 96-well plate (Maxisorp, Nunc Co., Rochester, NY). Alkaline phosphatase-conjugated goat anti-mouse Ig isotype and IgG subclass (Southern Biotechnology Associates Inc., Birmingham, Ala), and p-nitrophenyl phosphate (phosphatase substrate) were used to assay titers.
One way analysis of variance (ANOVA) procedures were used to assess significant differences among treatment groups. The Newman-Keuls test was used for comparisons of multiple group means.
Ab: antibody; GPCRs: G-Protein-coupled receptors; G-proteins: GTP-binding proteins; OVA: ovalbumin; LPS: lipopolysaccharide; TNP: trinitrophenyl; Ig: immunoglobulin; S1P: sphingosine 1-phosphate; WT: wild type; KO: knockout.
The authors declare that they have no competing interests.
SJL, CHL, SGK designed research; SJL and WHL performed research; SJL and WHL analyzed data; and CHL and SGK wrote the paper. All authors read and approved of the final manuscript.