Porcine Ig isotypes: function and molecular characteristics
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 IgGa is the predominant isotype produced in response to Streptococcus equi [6]. Horses immunized with Rhodococcus equi antigens in aluminium hydroxide produced an IgGb- and IgGT-biased response [7]. Aluminium hydroxide induces Type 2-biased immune response in mice, humans and other species [8]. Thus, equine IgGT and IgGb are putative Type 2 isotypes while IgGa 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 IgG2a is associated with decreased estimated breeding value (EBV) for clinical mastitis while IgG2b and IgG1 are positively correlated with increased EBV for mastitis [12]. Complement is efficiently activated by bovine allotype IgG2b and only half as well by IgG2a, suggesting that IgG2b 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, IgG2a, IgG2b, 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 IgG2a has a more flexible hinge than IgG2b, 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.
References (39)
- et al.
Complement-immunoglobulin interactions
Curr. Opin. Immunol.
(1995) - et al.
Anti-haemolysin IgG1 to IgG2 ratios correlate with haemolysin neutralization titres and lung lesion scores in Actinobacillus pleuropneumoniae infected pigs
Vaccine
(1998) - et al.
Serum and mucosal antibody isotype responses to M-like protein (SeM) of Streptpococcus equi in convalescent and vaccinated horses
Vet. Immunol. Immunopathol.
(1997) - et al.
Opsonization of Staphylococcus aureus by bovine immunoglobulin isotypes
J. Dairy Sci.
(1993) - et al.
IFN-γ stimulates IgG2 production from bovine B-cells co-stimulated with anti-μ and mitogen
Cell Immunol.
(1994) - et al.
Differential complement activation by bovine IgG2 allotypes
Vet. Immmunol. Immunopathol.
(1999) - et al.
Immunglobulin flexibility in complement activation
Immunol. Today
(1986) - et al.
CD20 positive human B lymphocytes separated with the magnetic cell sorter (MACS) can be induced to proliferation and antibody secretion in vitro
J. Immunol. Methods
(1989) - et al.
Characterization of antigen-specific receptors from pig lymphocytes
Mol. Immunol.
(1982) Bovine immunoglobulins: an augmented review
Vet. Immunol. Immunopathol.
(1983)
Bovine IgM: does it fix guinea pig complement in the absence of bovine complement components
Vet. Immunol. Immunopathol.
Molecular definition of interaction sites on human IgG for Fc receptors (huFc gamma R
Mol. Immunol.
Heterogeneity of bovine IgG2. VI. Comparative specificity of monoclonal and polyclonal capture antibodies for IgG2 (A1) and IgG2 (A2)
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.
Cited by (53)
Effects of oil-based adjuvants on the immune response of pigs after dermal administration of antigen and evaluation of the immunization level after a subsequent Actinobacillus pleuropneumoniae challenge in pigs
2023, Veterinary MicrobiologyCitation Excerpt :That might explain the effective level of protection against the APP infection induced by i.d. route of vaccine delivery. Similarly to the study Crawley et al. (2003) which reports, that protection against the toxigenic extra-cellular bacterium APP was correlated with an increased IgG(1): IgG(2) ratio of haemolytic toxin-specific antibodies. Porcine IgG1 and IgG2 isotypes exhibit close function properties, such as binding and neutralizing activity, ability to activate complement, trigger cytotoxicity and phagocytosis by immune cells (Paudyal et al., 2022).
T-cell factor-4 and MHC upregulation in pigs receiving a live attenuated classical swine fever virus (CSFV) vaccine strain with interferon-gamma adjuvant
2016, Veterinary JournalCitation Excerpt :This is consistent with previous studies indicating that cell-mediated immunity, in addition to antibody responses, could be important for protection against CSFV infection (Suradhat et al., 2001; Ganges et al., 2005). Alternatively, the IgG2 isotype showed better complement-activating activity than IgG1 in pigs (Crawley and Wilkie, 2003) and this may have contributed to viral eradication. Using several techniques, we established that Tcf-4 mRNA and protein expression were upregulated in the spleens of vaccinated pigs receiving IFN-γ, particularly in the PALS and the neighbouring area.
Full protection of swine against foot-and-mouth disease by a bivalent B-cell epitope dendrimer peptide
2016, Antiviral ResearchCitation Excerpt :Here, higher IgG2 titers for B2T(mal) vs. thioether-type platforms, particularly B4T(thio), is observed. Assuming that porcine IgG2 levels increase in response to cytokines such as IFN-γ and IL-12 (Crawley and Wilkie, 2003), our results suggest association between this Th1-biased isotype balance and improved protection. Opsonization seems to be an important mechanism for FMDV protection (Lannes et al., 2012; McCullough et al., 1988) and IgG2 isotype opsonizes and activates complement more efficiently than IgG1 (Bastida-Corcuera et al., 1999; Crawley and Wilkie, 2003).
Characterization of the immune response and evaluation of the protective capacity of rSsnA against Streptococcus suis infection in pigs
2016, Comparative Immunology, Microbiology and Infectious Diseases