Porcine Ig isotypes: function and molecular characteristics

doi:10.1016/S0264-410X(03)00142-7

Vaccine

Volume 21, Issues 21–22, 20 June 2003, Pages 2911-2922

https://doi.org/10.1016/S0264-410X(03)00142-7 Get rights and content

Abstract

In pigs, protection against the toxigenic extra-cellular bacterium Actinobacillus pleuropneumoniae was correlated with an increased IgG₁:IgG₂ ratio of haemolytic toxin-specific antibodies. In all species so far studied, IgG isotype expression is controlled by Type 1 (IFN-γ, IL-12) and Type 2 (IL-4, IL-10) cytokines which dictate immune response polarization to cell-mediated (CMI) or antibody-mediated immunity (AMI), respectively. Thus, immunoglobulin (Ig) isotypes reflect Type 1 or Type 2 immune responses. Immunoglobulin isotype production by porcine B-cells cultured in the presence of recombinant porcine (rp) cytokines varies by individual, however pigs tend to generate a high IgG₁:IgG₂ ratio in response to rp IL-10 and the inverse in response to rp IFN-γ or rp IL-12. Differential Ig isotype production should favor an isotype with a functional advantage to control the inciting infection and disease. However, functions of porcine Ig isotypes have not been described. To compare function of porcine IgM, IgG₁ and IgG₂ of defined specificity for hen eggwhite lysozyme (HEWL), Ig isotypes were affinity purified from serum by HEWL specificity and by isotype-specific mouse monoclonal antibodies. Their ability to activate complement (C′) and to opsonize was tested in vitro. Porcine IgG₂ had greater guinea pig C′ activating ability than did IgG₁. Neither isotype opsonized HEWL-conjugated sheep erythrocytes in vitro. Amino acid sequence analysis of IgG isotypes revealed that all subclasses have putative C′ binding sites but that IgG_2a, IgG_2b and IgG₄ were more flexible in the middle hinge region than IgG₁ and IgG₃ and would likely activate C′ more efficiently. Thus, porcine IgG isotypes associated with resistance and susceptibility to disease also differ in their actual and predicted biological functions.

Introduction

Differential immunoglobulin (Ig) isotype expression permits diverse isotype-related functions, appropriate to the inducing infection or immunization, such as complement (C′) activation and binding to Fc receptors on phagocytic cells [1]. Production of Ig isotypes is controlled by Type 1 (IFN-γ, IL-12) and Type 2 (IL-4, IL-10) cytokines which polarize response to cell-mediated (CMI) or antibody-mediated immunity (AMI) immune response. Typically, C′ and phagocytosis-activating Ig isotypes are induced by Type 1 cytokines.

Porcine immunoglobulin (Ig) isotypes have been related to resistance and susceptibility to infection, but isotype-related function has not been described. The ratio of IgG₁:IgG₂-associated antibody was increased as a correlate of resistance to the toxigenic extra-cellular pathogen Actinobacillus pleuropneumoniae suggesting Type1/Type2 control of Ig isotype switching and differential function of pig IgG₁ and IgG₂ such that IgG₁ is hypothetically a Type 2 isotype and IgG₂ Type 1 [2]. Pig cytokines regulate Ig isotype expression, since recombinant porcine (rp) IFN-γ or rp IL-12 up-regulate IgG₂ production by enriched porcine B-cells cultured in vitro, while rp IL-10 up-regulates IgG₁ production, although results varied among individuals [3].

Differential Ig isotype expression is reported in several species. Horses respond to intestinal nematodes, with a strong IgGT response [4], [5] while IgG_a is the predominant isotype produced in response to Streptococcus equi [6]. Horses immunized with Rhodococcus equi antigens in aluminium hydroxide produced an IgG_b- and IgGT-biased response [7]. Aluminium hydroxide induces Type 2-biased immune response in mice, humans and other species [8]. Thus, equine IgGT and IgG_b are putative Type 2 isotypes while IgG_a is Type 1. Human IgG₁ and IgG₃ are efficient potentiators of antibody-dependent cell-mediated cytotoxicity, opsonization and phagocytosis associated with protection from severe malaria [9]. In contrast, IgG₂ inhibits this protection. Opsonization of Staphylococcus aureus, the major cause of mastitis in cows, is mediated by IgG₂, a Type 1-associated isotype, and inhibited by IgG₁ [10], [11]. The bovine allotype IgG_2a is associated with decreased estimated breeding value (EBV) for clinical mastitis while IgG_2b and IgG₁ are positively correlated with increased EBV for mastitis [12]. Complement is efficiently activated by bovine allotype IgG_2b and only half as well by IgG_2a, suggesting that IgG_2b is important in killing, inactivating or opsonizing protozoa, bacteria and viruses [13].

Variation in protein structure determines functional differences among Ig isotypes. Five distinct amino acid sequences are described for porcine IgG isotypes, grouped according to sequence similarity into two clusters. One cluster comprises IgG₁ and IgG₃ and the other, IgG_2a, IgG_2b, and IgG₄ [14]. Porcine IgG isotypes have similar constant region sequences, with subclass differences due primarily to variation in hinge and constant heavy chain 3 (CH3) regions. In other species, the length and flexibility of the hinge which is subdivided into upper, middle and lower hinge regions [15], vary by IgG subclass and species and are associated with the initiation of the classical C′ cascade [16]. The upper hinge region provides flexibility between the Fab arms and rotational motion for each Fab. The rigid poly-proline middle hinge, stabilized by interchain cysteines forming disulphide bridges, acts as a spacer between the Fab arms and Fc sites involved in binding the first component of the C′ cascade, C1q and is less flexible while the lower hinge is more flexible [17]. Hinges are rendered rigid by large, hydrophobic amino acids, positioning the Fab arms so as to interfere with C1q binding. Small, hydrophilic amino acids generate more flexible structures. Bovine IgG_2a has a more flexible hinge than IgG_2b, providing a possible explanation for its superior ability to activate C′ [13]. Hinge flexibilities have not been described for porcine IgG isotypes, thus a physical–chemical analysis of neighbouring amino acid properties [18] was undertaken as an adjunct to direct investigation of isotype functions. The research reported here tests the hypothesis that porcine IgG isotypes differ functionally in protection as a correlate of reported isotype bias. Results provide possible design objectives for future vaccines or adjuvants as well as criteria for detecting Type 1 and Type 2 immune response bias in pigs.

Section snippets

Experimental design

The present study was designed to investigate biological functions of porcine immunoglobulin (Ig) isotypes, and to predict secondary structures that may influence these functions. Complement (C′) activation of affinity-purified hen eggwhite lysozyme (HEWL)-specific porcine IgM, IgG₁ and IgG₂ was assessed by measuring their lysis of HEWL-sensitized sheep red blood cells (sRBC) in the presence of C′. Opsonic activity of the purified Igs was described as the phagocytic index of porcine macrophage

Molecular analysis of affinity-purified immunoglobulins

Estimates of the molecular sizes of affinity-purified porcine IgM, IgG₁, and IgG₂ heavy and light chains were determined by electrophoresis in a 12% polyacrylamide gel after reduction in 2-mercaptoethanol and denaturation with SDS (Fig. 1A). All IgG isotypes had heavy and light chains of approximately 50–55 and 22–25 kDa, respectively. A band at 80 kDa, representing IgM heavy chain, was also present in all samples. Non-reducing PAGE indicated that affinity-purified porcine IgM, IgG₁, and IgG₂

Discussion

The abilities of porcine IgM, IgG₁ and IgG₂ to activate heterologous C′ and to opsonize targets were studied as possible correlates of in vivo functions. The investigation was provoked by apparent importance of the IgG₁:IgG₂ ratio of haemolysin-specific antibody in resistance and susceptibility of pigs to A. pleuropneumoniae-induced disease which suggested Type 2:Type 1 control of immune response, and possibly an isotype functional bias [2].

Porcine IgG isotypes, affinity-purified with

Acknowledgements

This research was supported by a grant to B.N. Wilkie from the Natural Sciences and Engineering Research Council of Canada. We thank Dr. K. Nielsen for providing monoclonal antibodies.

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Porcine Ig isotypes: function and molecular characteristics

Abstract

Introduction

Section snippets

Experimental design

Molecular analysis of affinity-purified immunoglobulins

Discussion

Acknowledgements

Curr. Opin. Immunol.

Vaccine

Vet. Immunol. Immunopathol.

J. Dairy Sci.

Cell Immunol.

Vet. Immmunol. Immunopathol.

Immunol. Today

J. Immunol. Methods

Mol. Immunol.

Vet. Immunol. Immunopathol.

Vet. Immunol. Immunopathol.

Mol. Immunol.

Vet. Immunol. Immunopathol.

Differential control of immunoglobulin isotype production by porcine B-cells cultured with cytokines

Vet. Immunol. Immunopathol.

Increase in immunoglobulin T in ponies as a response to experimental infection with the nematode Stongylus vulgaris

Am. J. Vet. Res.

Correlation of antigen specific IgG and IgG(T) responses with Anoplocephal perfoliata infection intensity in the horse

Parasite Immunol.

Use of Rhodococcus equi virulence-associated protein for immunization of foals against Rhodococcus equi pneumonia

Am. J. Vet. Res.

Aluminium hydroxide adjuvant initiates strong antigen-specific responses in the absence of IL-4 or IL-13-mediated signalling

J. Immunol.

Naturally acquired immunoglobulin (Ig)G subclass antibodies to crude asexual Plasmodium falciparum lysates: evidence for association with protection for IgG1 and disease for IgG2

Parasite Immunol.

Naturally acquired immunoglobulin (Ig)G subclass antibodies to crude asexual Plasmodium falciparum lysates: evidence for association with protection for IgG₁ and disease for IgG₂