Understanding the vertebrate immune system: insights from the reptilian perspective.

Reptiles are ectothermic amniotes, providing the key link between ectothermic anamniotic fishes and amphibians, and endothermic amniotic birds and mammals. A greater understanding of reptilian immunity will provide important insights into the evolutionary history of vertebrate immunity as well as the growing field of eco-immunology. Like mammals, reptile immunity is complex and involves innate, cell-mediated and humoral compartments but, overall, there is considerably less known about immune function in reptiles. We review the current literature on each branch of the reptilian immune system, placing this information in context to other vertebrates. Further, we identify key areas that are prime for research as well as areas that are lagging because of lack of reagents in non-model systems.

Lovesick: immunological costs of mating to male sagebrush crickets.

A growing body of evidence suggests that resources invested in reproduction often come at the expense of the ability to mount an immune response. During mating, female sagebrush crickets, Cyphoderris strepitans, consume the ends of the male's hind wings and ingest his haemolymph. Previous research has shown that this behaviour impairs the ability of males to secure additional matings. One hypothesis to account for this effect is that wing wounding triggers an energetically costly immune response, such that nonvirgin males are unable to sustain the costly acoustical signalling needed to attract additional females. To test this hypothesis, we injected virgin males with lipopolysaccharides (LPS) to provoke an immune response, and monitored their mating success in the field. LPS-injected virgin males took significantly longer to mate than sham-injected virgin males, and spent significantly less time calling. We also compared virgin, nonvirgin and experimentally wing-wounded virgin males with respect to: (1) their ability to encapsulate a foreign invader via the accumulation of haemocytes and deposition of melanin and (2) baseline levels of phenoloxidase (PO), a key enzyme in the biochemical cascade leading to the production of melanin. Although encapsulation ability did not differ with reproductive experience, virgin males had significantly higher levels of PO than either nonvirgin or experimentally wing-wounded virgin males. These results suggest that wing-wounding alone is sufficient to impair male immunity, and that males trade-off investment in reproduction and immunity.

Immune-challenged house wren broods differ in the relative strengths of their responses among different axes of the immune system.

Single components of the immune system are widely used to assess immune function in free-living vertebrates. However, as different immunological components are triggered by different types of threats and may be regulated independently, there is little reason to assume that they should respond similarly if challenged. We investigated whether three commonly assayed immune responses, cutaneous immune activity (phytohaemagglutinin assay), antibody response (tetanus toxoid immunization), and plasma bactericidal activity (Escherichia coli killing) are positively related in nestling house wrens (Troglodytes aedon). Multivariate analysis revealed significant differences in overall immune responsiveness among broods (i.e. nests), primarily attributable to differences in plasma bactericidal activity. Among broods, humoral immune response was negatively related to cutaneous immune activity and positively related to plasma bactericidal activity. We found no significant relationships among these measures of immunity among individual nestlings within broods. Our results suggest that different broods (i.e. families) invest differentially in the various branches of the immune system. Further study is needed to characterize the roles of maternal, genetic and environmental effects on the expression of this physiological bias.

B cells of aged mice show decreased expansion in response to antigen, but are normal in effector function.

Increased dysfunction of the immune system with age can be attributed to developmental changes in cell types critical for proper immune responses. Previous studies have shown defects in humoral responses of aged individuals, but have not distinguished between aged T-cell/microenvironment and intrinsic B-cell defects. Here adoptive transfer of antigen-specific transgenic B cells compared early immunopoeisis from young and aged donors in a young recipient environment. B cells from aged donors demonstrated decreased antigen-induced expansion, particularly in the lymph nodes; however, they acquired a germinal center phenotype at frequencies similar to B cells from young donors. Additionally, aged B cells produced equivalent levels of antigen-specific antibody that underwent affinity maturation and isotype switching and demonstrated similar numbers of antibody-secreting cells of switched isotype. Thus, the ability of aged B cells to respond appropriately to T-dependent antigens and differentiate into high-affinity, isotype-switched, antibody-secreting cells appears to be intact.

B cell immunopoiesis: visualizing the impact of CD40 engagement on the course of T cell-independent immune responses in an Ig transgenic system.

This study tracks the fate of antigen-reactive B cells through follicular and extrafollicular responses and addresses the function of CD40 in these processes. The unique feature of this system is the use of transgenic B cells in which the heavy chain locus has been altered by site-directed insertion of a rearranged V(H) DJ(H) exon such that they are able to clonally expand, isotype-switch and follow a normal course of differentiation upon immunization. These Ig transgenic B cells when adoptively transferred into non-transgenic (Tg) mice in measured amounts expanded and differentiated distinctively in response to T cell-independent (TI) or T cell-dependent (TD) antigens. The capacity of these Tg B cells to faithfully recapitulate the humoral immune response to TI and TD antigens provides the means to track clonal B cell behavior in vivo. Challenge with TI antigen in the presence of agonistic anti-CD40 mAb resulted in well-defined alterations of the TI response. In vivo triggering of Tg B cells with TI antigen and CD40 caused an increase in the levels IgG produced and a broadening of the Ig isotype profile, characteristics which partially mimic TD responses. Although some TD characteristics were induced by TI antigen and CD40 triggering, the Tg B cells failed to acquire a germinal center phenotype and failed to generate a memory response. Therefore, TD-like immunity can be only partially reconstituted with CD40 agonists and TI antigens, suggesting that there are additional signals required for germinal center formation and development of memory.

CD40 and its crucial role as a member of the TNFR family.

The cell surface molecule CD40 is a member of the tumor necrosis factor receptor (TNFR) family and plays a pivotal role in the immune system due in part to its wide tissue distribution. Early studies focused on CD40 in B cell biology, but recent work demonstrates a broader role for CD40 in the immune system contributing to cell-mediated as well as humoral immune responses. Disruption of CD40 and its ligand, CD154 leads to immunodeficiency as in the case of hyper IgM syndrome. Likewise, blockade of CD40-CD154 interactions can be exploited to control misguided autoimmune responses. This article provides a brief overview of CD40 with a focus on its involvement in disease and its usefulness as a therapeutic target.

Direct binding of IL-12 to human and murine B lymphocytes.

IL-12 has been shown to play a central role in cell-mediated inflammatory reactions through direct activation of T cells and NK cells. IL-12 also strongly influences humoral immunity but these effects have been thought to be indirect and caused by intermediary cytokines. Using flow cytometry, we now show that IL-12 directly interacts with B cells. Freshly isolated murine peritoneal B-1 and conventional B lymphocytes bound IL-12, but splenic B cells failed to react unless first stimulated with lipopolysaccharide. All murine B cell sources were found to express IL-12R beta 1 subunit transcripts as detected by PCR and RNase protection assays. IL-12 binding was also detected on phytohemagglutinin-stimulated human T cell blasts and Staphylococcus aureusl IL-2-stimulated B cell blasts but not on freshly isolated peripheral blood lymphocytes. Similarly, IL-12 directly bound to the human SKW6.4 Burkitt's B cell lymphoma line. In all cases positive staining was ablated by omitting IL-12 from the procedure, showing that it was not due to detection of endogenous IL-12. These findings indicate that B cells represent another major target for IL-12 in addition to T and NK cells, and that IL-12 can directly affect humoral immunity.

Enhancement of humoral immunity by interleukin-12.

We have found that IL-12 treatment of mice leads to long-lasting enhancement in production of most antibody isotypes in conventional B-cell responses. Initial recruitment of new B-cell clones into the response is mediated by IFN-gamma, but subsequent enhancement of Ig secretion appears to be IFN-gamma-independent. We have further found that activated B cells can directly bind IL-12. Taken together, our results suggest a two-step model for the role of IL-12 in enhancement of humoral immunity. Initially, IL-12 induces production of IFN-gamma from Th1 and NK cells. Enough cytokine can be produced from either cell type to then mediate gamma 2a heavy chain isotype switching as well as temporary suppression of IgG1 production. IL-12 further stimulates post-switched cells, including cells producing IgG1, to secrete greatly increased amounts of antibody. This step is not mediated by IFN-gamma but might be due to direct IL-12 binding to activated B lymphocytes. Depletion of B1 cells by IL-12 may further enhance antibody responsiveness since B1 cells are known to competitively inhibit Ig secretion by conventional B cells. The end result is that IL-12 causes a generalized upregulation in production of all antibodies and therefore acts as a strong adjuvant for humoral as well as cellular immunity.

Inhibition of murine B1 lymphocytes by interleukin-12.

B1 cells are a subset of B lymphocytes found in many species and are implicated in the development of autoimmunity. B1 cells have previously been shown to be suppressed by the T helper (Th)1 cytokine interferon (IFN)-gamma, and to be stimulated by the Th2 cytokines interleukin (IL)-2, IL-4, IL-5 and IL-10. To examine further the interactions of B1 cells and Th1 cells, we have now tested the effects of the Th1 cell-inducing cytokine IL-12 on murine B1 cells. BALB/c mice were immunized with phosphorylcholine conjugated to keyhole limpet hemocyanin (PC-KLH) and simultaneously treated with 1 microgram recombinant murine IL-12 for 3 consecutive days. In addition to altering the isotype and idiotype distribution of anti-PC antibodies, IL-12 treatment was found to cause a loss of peritoneal, but not splenic B lymphocytes in immunized mice. B cell depletion required exposure to IL-12 plus antigenic stimulation. Levels of peritoneal B lymphocytes were fully restored by day 45, but the majority of these cells belonged to the B2 subset. Additionally, proliferation of B1 cells in vitro induced by IL-5 was substantially inhibited by IL-12. IL-12 itself had no effect on viable cell recovery of peritoneal cells (PeC) cultured in vitro, but viable cell recovery was significantly decreased in PeC cultured with IL-5 plus IL-12. These results show that IL-12 causes the loss of murine peritoneal B1 cells and suggest that treatment with this cytokine may be useful for disease conditions that involve B1 cell dysfunction.

Interleukin 12 alters the isotype-restricted antibody response of mice to hen eggwhite lysozyme.

Protein antigens elicit humoral responses in mice that consist predominantly of IgG1 antibodies. We have now investigated the ability of IL-12, a cytokine reported to augment IgG2a anti-hapten responses through activation of Th1 cells, to alter antibody responses to hen eggwhite lysozyme (HEL). The normal response of BALB/c mice to HEL is highly restricted to IgG1 expression and therefore provides an excellent system for determining effects of cytokines on expression of other isotypes. Seven days after immunization, IL-12 treated mice demonstrated greatly elevated HEL-specific IgG2a antibody levels and suppressed IgG1 production, while PBS-treated control mice showed a typical IgG1-restricted response. On day 28, IL-12-treated mice showed heightened serum antibody levels of both isotypes. Delaying cytokine treatment until after the typical IgG1 anti-HEL response had already been established also led to significant elevation of serum IgG2a antibody levels. These effects correlated with increased IFN-gamma production; however, administration of IL-12 plus anti-IFN-gamma had little influence on IgG2a enhancement, although it did relieve the early IgG1 suppression. Furthermore, the differential effects of Il-12 on isotype expression did not correlate with time; in fact, IgG2a enhancement correlated with loss of IgG1 suppression. Our findings indicate that (i) IL-12 reproducibly induces large amounts of IgG2a HEL-specific antibodies in vivo; (ii) it can alter isotype profiles of both primary and secondary responses; and (iii) its effects on humoral immunity are not completely explained by induction of Th1 cell derived IFN-gamma.

Antibody response of murine B1 cells to hen eggwhite lysozyme.

Cell transfer studies were performed to investigate the ability of murine peritoneal B1 cells to produce specific IgG antibody against the T-dependent protein antigen, hen eggwhite lysozyme (HEL). Peritoneal cells (PeC) from normal BALB/c mice were transferred into newborn, allotype-congenic, C.B-17 severe combined immunodeficient (SCID) mice alone or together with splenic T cells from HEL-primed C.B-17 mice. After immunization with HEL, only those mice that received both PeC and primed T cells produced HEL-specific IgG of the PeC donor allotype. To identify the B cell subset responsible for antibody production, PeC were sorted before transfer into B1 and conventional B (B2) cell populations. It was found that transfer of purified B1 cells plus primed T cells resulted in high levels of IgG1 anti-HEL in approximately half of the SCID recipients, while mice receiving B2 cells produced little detectable antibody. The responses consisted primarily of IgG1 kappa anti-HEL, with no significant IgM or lambda-bearing components. Seventeen HEL-specific hybridomas of BALB/c origin, i.e., derived from the B1 cell donor, were obtained from reconstituted SCID mice after various periods of immunization and analyzed for fine specificity using a panel of avian lysozymes. All but one of the B1 cell-derived mAbs recognized an HEL epitope not present on the closely related bobwhite quail lysozyme (differing from HEL at only 4 of 129 amino acid positions). While IEF analyses demonstrated the presence of extensive clonotypic diversity, the epitope specificity pattern, which is rare among B2-cell-derived antibodies, suggests that the B1 cell recognition repertoire for HEL is severely limited.