Functional effects of CCL3L1 copy number.

Copy number variation (CNV) is becoming increasingly important as a feature of human variation in disease susceptibility studies. However, the consequences of CNV are not so well understood. Here, we present data exploring the functional consequences of CNV of CCL3L1 in 55 independent UK samples with no known clinical phenotypes. The copy number of CCL3L1 was determined by the paralogue ratio test, and expression levels of macrophage inflammatory protein-1α (MIP-1α) and mRNA from stimulated monocytes were measured and analysed. The data show no statistically significant association of MIP-1α protein levels with copy number. However, there was a significant correlation between copy number and CCL3L1:CCL3 mRNA ratio. The data also provide evidence that expression of CCL3 predominates in both protein and mRNA, and therefore the observed variation of CCL3 is potentially more important biologically than that of CNV of CCL3L1.

Fc-receptors and immunity to malaria: from models to vaccines.

The complexity and number of antigens (Ags) seen during an immune response has hampered the development of malaria vaccines. Antibodies (Abs) play an important role in immunity to malaria and their passive administration is effective at controlling the disease. Abs represent approximately 25% of all proteins undergoing clinical trials, and these 'smart biologicals' have undergone a major revival with the realization that Abs lie at the interface between innate and adaptive immunity. At least 18 Abs have FDA approval for clinical use and approximately 150 are in clinical trials, the majority for the treatment of cancer, allograft rejection or autoimmune disease. Despite these triumphs none are in development for malaria, principally because they are perceived as being too expensive for a disease mainly afflicting poor and marginalized populations. Although unlikely, at least in the foreseeable future, that Ab-based prophylaxis will be made available to the millions of people at risk from malaria, they may be incorporated into current vaccine approaches, since Abs act as correlates of protection in studies aimed at defining the best Ags to include in vaccines. Abs may also form the basis for novel vaccination strategies by targeting Ags to appropriate antigen presenting cells. Therefore, to develop the most efficacious vaccines it will be necessary to fully understand which Abs and Fc-receptors (FcRs) are best engaged for a positive outcome.

When is a malaria immune complex not an immune complex?

Immune complexes (ICs) are believed to play an important role in malaria pathology, and an interesting article by Mibei et al. recently published by Parasite Immunology suggests that IgG4 and IgE are particularly important. However, researchers should be aware of potential pitfalls to current assays aimed at measuring plasma ICs and correlating these to deposition in tissues.

Characterization of immunoglobulin binding by schistosomes.

Although controversial, schistosomes are believed to cloak themselves in antibody through non-specific interactions with the immunoglobulin (Ig) molecule. The acquisition of host Ig by the schistosome may mask its foreign status and/or interfere with Fc-dependent functions. We report experiments aimed at characterizing the interaction between Ig-Fc and paramyosin, a schistosome Fc-receptor previously reported to bind human IgG. We show that certain Ig classes, in particular murine IgG2b and IgG3, are not only able to bind recombinant paramyosin, but also associate with other parasite proteins. The Fc region of IgG contains four hydrophobic patches, two of which are known to interact with distinct molecules: one in the Cgamma2-Cgamma3 interdomain region bound by protein G, mannose binding lectin (MBL), and the neonatal Fc-receptor (FcRn), and one at the top of the Cgamma2 domain bound by phagocytic FcgammaRs and C1q. We provisionally discounted the involvement of these regions, since IgG binding by paramyosin did not inhibit FcgammaR-mediated NADPH respiratory bursts, and protein G was unable to block IgG binding to paramyosin. Given their apparent low affinity, we postulate hydrogen bonding between reactive residues in a hydrophobic patch at the bottom of the Cgamma3 domain and negatively charged Glu or Asp amino acids in paramyosin.

Heterogeneous interspecific interactions in a host-parasite system.

Macroparasites of vertebrates usually occur in multi-species communities, producing infections whose outcome in individual hosts or host populations may depend on the dynamics of interactions amongst the different component species. Within a single co-infection, competition can occur between conspecific and heterospecific parasite individuals, either directly or via the host's physiological and immune responses. We studied a natural single-host, multi-parasite model infection system (polystomes in the anuran Xenopus laevis victorianus) in which the parasite species show total interspecific competitive exclusion as adults in host individuals. Multi-species infection experiments indicated that competitive outcomes were dependent on infection species composition and strongly influenced by the intraspecific genetic identity of the interacting organisms. Our results also demonstrate the special importance of temporal heterogeneity (the sequence of infection by different species) in competition and co-existence between parasite species and predict that developmental plasticity in inferior competitors, and the induction of species-specific host resistance, will partition the within-host-individual habitat over time. We emphasise that such local (within-host) context-dependent processes are likely to be a fundamental determinant of population dynamics in multi-species parasite assemblages.

Streptococcal IgA-binding proteins bind in the Calpha 2-Calpha 3 interdomain region and inhibit binding of IgA to human CD89.

Certain pathogenic bacteria express surface proteins that bind to the Fc part of human IgA or IgG. These bacterial proteins are important as immunochemical tools and model systems, but their biological function is still unclear. Here, we describe studies of three streptococcal proteins that bind IgA: the Sir22 and Arp4 proteins of Streptococcus pyogenes and the unrelated beta protein of group B streptococcus. Analysis of IgA domain swap and point mutants indicated that two loops at the Calpha2/Calpha3 domain interface are critical for binding of the streptococcal proteins. This region is also used in binding the human IgA receptor CD89, an important mediator of IgA effector function. In agreement with this finding, the three IgA-binding proteins and a 50-residue IgA-binding peptide derived from Sir22 blocked the ability of IgA to bind CD89. Further, the Arp4 protein inhibited the ability of IgA to trigger a neutrophil respiratory burst via CD89. Thus, we have identified residues on IgA-Fc that play a key role in binding of different streptococcal IgA-binding proteins, and we have identified a mechanism by which a bacterial IgA-binding protein may interfere with IgA effector function.

Fc receptors and immunity to parasites.

Fc receptors (FcRs) are crucial in the immune system; they mediate a plethora of biological functions as diverse as antigen presentation, phagocytosis, cytotoxicity, induction of inflammatory cascades and modulation of immune responses. Parasites, in order to survive in the immunocompetent host, have devised ingenious methods to subvert this important aspect of the immune response. This article discusses the current thinking on FcRs, their role in immunity to parasites, and immune evasion strategies employed by parasites in their attempt to neutralize the important immune defense mechanisms mediated by these molecules.

Cleavage of human IgE mediated by Schistosoma mansoni.

BACKGROUND: IgE-mediated mechanisms are important in protection against helminth parasites. However, schistosomes are long-lived in mammalian hosts, presumably as a result of immune evasion strategies. We sought evidence for one such strategy, namely specific cleavage of host IgE. METHODS: Human IgE, IgA and IgG were incubated with extracts from cercarial and schistosomular stages of Schistosoma mansoni or with schistosomular culture supernatants. The resulting products were analysed by Western blotting with Ig-specific antibodies. Numerous protease inhibitors were assessed for ability to inhibit the observed cleavage of IgE by the extracts. Partial purification of the IgE-proteolytic activity from cercarial extract was achieved by gel filtration. To test IgE function, we compared the abilities of untreated and schistosomular-treated IgE to mediate rosette formation through interaction with Fcepsilon receptors. RESULTS: Cercarial and schistosomular extracts were found to cleave human, mouse and rat IgE but not human IgA1, IgA2 or IgG1. Schistosomular culture supernatants displayed similar proteolytic activity towards IgE. Immunoblotting suggested that cleavage occurred close to the Cepsilon2/Cepsilon3 domain interface of the IgE heavy chain. PMSF and elastatinal inhibited cleavage, suggesting that the protease involved is an elastase-like serine protease, particularly since porcine pancreatic elastase also cleaved IgE to give similar-sized products. Further, the chloromethyl ketone derivatized peptide MeO-Suc-Ala-Ala-Pro-Leu- CMK, known to specifically inhibit the schistosome elastase, prevented IgE cleavage. Cleavage of human IgE rendered the antibody molecule unable to interact with U937 cells expressing FcepsilonRII. CONCLUSIONS: An elastase-like protease in S. mansoni is able to render IgE non-functional.

Identification of residues in the CH2/CH3 domain interface of IgA essential for interaction with the human fcalpha receptor (FcalphaR) CD89.

Cellular receptors for IgA (FcalphaR) mediate important protective functions. An extensive panel of site-directed mutant IgAs was used to identify IgA residues critical for FcalphaR (CD89) binding and triggering. Although a tailpiece-deleted IgA1 was able to bind and trigger CD89, antibodies featuring CH3 domain exchanges between human IgA1 and IgG1 could not, indicating that both domains but not the tailpiece are required for FcalphaR recognition. To further investigate the role of the interdomain region, numerous IgA1s, each with a point substitution in either of two interdomain loops (Leu-257-Gly-259 in Calpha2; Pro-440-Phe-443 in Calpha3), were generated. With only one exception (G259R), substitutions produced either ablation (L257R, P440A, A442R, F443R) or marked reduction (P440R) in CD89 binding and triggering. Further support for involvement of these interdomain loops was provided by interspecies comparisons of IgA. Thus a human IgA1 mutant, LA441-442MN, which mimicked the mouse IgA loop sequence through substitution of two adjacent residues in the Calpha3 loop, was found, like mouse IgA, not to bind CD89. In contrast, bovine IgA1, identical to human IgA1 within these interdomain loops despite numerous differences elsewhere in the Fc region, did bind CD89. We have thus identified motifs in the interdomain region of IgA Fc critical for FcalphaR binding and triggering, significantly enhancing present understanding of the molecular basis of the IgA-FcalphaR interaction.

The glycosylation and structure of human serum IgA1, Fab, and Fc regions and the role of N-glycosylation on Fcα receptor interactions.

The human serum immunoglobulins IgG and IgA1 are produced in bone marrow and both interact with specific cellular receptors that mediate biological events. In contrast to IgA1, the glycosylation of IgG has been well characterized, and its interaction with various Fc receptors (Fc Rs) has been well studied. In this paper, we have analyzed the glycosylation of IgA1 and IgA1 Fab and Fc as well as three recombinant IgA1 molecules, including two N-glycosylation mutants. Amino acid sequencing data of the IgA1 Fc O-glycosylated hinge region indicated that O-glycans are located at Thr228, Ser230, and Ser232, while O-glycan sites at Thr225 and Thr236 are partially occupied. Over 90% of the N-glycans in IgA1 were sialylated, in contrast to IgG, where < 10% contain sialic acid. This paper contains the first report of Fab glycosylation in IgA1, and (in contrast to IgG Fab, which contains only N-linked glycans) both N- and O-linked oligosaccharides were identified. Analysis of the N-glycans attached to recombinant IgA1 indicated that the Cα 2 N-glycosylation site contained mostly biantennary glycans, while the tailpiece site, absent in IgG, contained mostly triantennary structures. Further analysis of these data suggested that processing at one Fc N-glycosylation site affects the other. Neutrophil Fcα R binding studies, using recombinant IgA1, indicated that neither the tailpiece region nor the N-glycans in the C alpha 2 domain contribute to IgA1-neutrophil Fcα R binding. This contrasts with IgG, where removal of the Fc N-glycans reduces binding to the Fcγ R. The primary sequence and disulfide bond pattern of IgA1, together with the crystal structures of IgG1 Fc and mouse IgA Fab and the glycan sequencing data, were used to generate a molecular model of IgA1. As a consequence of both the primary sequence and S-S bond pattern, the N-glycans in IgA1 Fc are not confined within the inter-α-chain space. The accessibility of the Cα 2 N-glycans provides an explanation for the increased sialylation and galactosylation of IgA1 Fc over that of IgG Fc N-glycans, which are confined in the space between the two Cγ 2 domains. This also suggests why in contrast to IgG Fc, the IgA1 N-glycans are not undergalactosylated in rheumatoid arthritis.

Structural requirements for assembly of dimeric IgA probed by site-directed mutagenesis of J chain and a cysteine residue of the alpha-chain CH2 domain.

The structural features of J chain required for interaction with IgA in IgA dimer assembly were investigated by coexpression of wild-type and mutant forms of J chain with IgA1 in CHO cells. With wild-type J chain, a mixture of J chain-containing dimers and monomers was secreted. Substitution of Cys14 of J chain with Ser resulted in expression of only monomer IgA covalently associated with J chain. Similarly, mutation of Cys68 to Ser also resulted in expression predominantly of a monomer IgA-J chain species. These results suggest that Cys14 and Cys68 play critical roles in formation of J chain-containing IgA dimers, with each forming a disulfide bridge to an IgA monomer. Substitution of Asn48 with Ala, to prevent attachment of N-linked carbohydrate to J chain, also resulted in markedly reduced dimer assembly, suggesting a requirement for the sugar moiety in J chain function. We also mutated Cys311 on the C alpha2 domain of the IgA heavy chain to Ser. When coexpressed with wild-type J chain, this mutant was still capable of forming dimers, indicating that this residue was not involved in dimerization. Taken together, our results are consistent with an arrangement in which IgA monomers are linked end-to-end with J chain interposed.

Alternative splicing of the human IgA Fc receptor CD89 in neutrophils and eosinophils.

Receptors for the Fc portion of IgA (Fc alpha R) trigger important immunological elimination processes against IgA-coated targets. Investigation of human Fc alpha R (CD89) transcripts in neutrophils, eosinophils and a monocyte-like cell line, THP-1, with the use of reverse transcriptase PCR, Northern blotting and RNase protection analysis, has provided evidence in these cell types for at least two distinct transcripts generated by alternative splicing. The cDNAs derived from the two major transcripts of both neutrophils and eosinophils have been cloned and sequenced. For both cell types, the larger clone represents the previously described full-length receptor, whereas the second, shorter, splice variant lacks the entire second, membrane-proximal, Ig-like domain. Stable CHO-K1 transfectants have been obtained for both full-length and truncated variant neutrophil receptors. Whereas the full-length receptor is recognized by a panel of five anti-Fc alpha R monoclonal antibodies (mAbs), the shorter variant is bound weakly by only two of the antibodies, suggesting that the epitopes recognized by the majority of the mAbs lie at least in part in the second Ig-like domain of Fc alpha R. Both full-length and splice variant forms of the receptor bind secretory IgA, but the weak binding to serum IgA seen with the full-length receptor is not evident with the shorter variant. Alternative splicing might therefore serve as a means of diversifying Fc alpha R structure and function.

Mutagenesis of the human IgA1 heavy chain tailpiece that prevents dimer assembly.

The structural features of the human IgA1 tailpiece required for interaction with J chain in IgA dimer assembly were investigated using a protein engineering approach. Wild-type and mutant forms of IgA1 were expressed in the mouse myeloma cell line, J558L, which endogenously expresses J chain. Wild-type IgA1 was secreted as a mixture of dimers and monomers. Deletion of the entire tailpiece by stop codon introduction completely prevented dimer formation. Similarly, substitution of the penultimate residue of the tailpiece, Cys471, with serine resulted in the secretion of IgA monomers alone. Substitution of Asn459 with alanine to prevent attachment of N-linked carbohydrate to the tailpiece also resulted in markedly reduced dimer assembly. These results indicate the critical role played by the tailpiece, and Cys471 in particular, in IgA dimerization. In addition, we found tailpiece-deleted IgA1 and the Cys to Ser471 mutant IgA1 were secreted as mixtures of covalently associated monomers (alpha 2L2) and alpha L half-molecules. The tailpiece may thus play some role in promoting the association of alpha-chains required for IgA monomer assembly.

The effect of gamma-radiation and heat shock on protein synthesis and antioxidant enzymes in the gastrointestinal parasite, Heligmosomoides polygyrus.

Protein synthesis and antioxidant enzyme activities were investigated in gamma-irradiated (300 Gy) and heat shocked (42 degrees C) larval stages of the gastrointestinal parasite, Heligmosomoides polygyrus bakeri (H. polygyrus). No qualitative or quantitative differences were observed in the incorporation of (35S)-methionine into somatic proteins of unirradiated or irradiated exsheathed third-stage (L3) larvae at either 37 degrees C or 42 degrees C. The rate of protein synthesis doubled in L3 stages maintained at 42 degrees C compared with 37 degrees C, irrespective of whether the larvae had been irradiated or not. The composition of excretory/secretory (ES) proteins varied between unirradiated and irradiated exsheathed L3 larvae maintained under identical conditions. Prominent heat-inducible proteins of 26 and 17 kDa were synthesised and excreted at 42 degrees C by both unirradiated and irradiated L3 stages. No major differences in protein synthesis could be detected between unirradiated and irradiated fourth-stage (L4) larvae. Temperature elevation significantly reduced protein synthesis in L4 stages, most notably in unirradiated parasites. Heat-inducible proteins were not detected in response to either irradiation or temperature elevation in L4 larvae. Immune sera recognised a similar spectrum of antigens in both unirradiated and irradiated L4 somatic and ES preparations and reacted with antigens from irradiated L4 parasites with less intensity than with antigens from unirradiated L4 larvae. Catalase was the only antioxidant enzyme examined with activity that changed significantly in irradiated parasites, being reduced to approximately 36% of normal levels in irradiated L4 stages. No significant difference existed between irradiated and unirradiated parasites in the levels of activity of superoxide dismutase and glutathione reductase.

Irradiated larval vaccination and antibody responses evaluated in relation to the expression of immunity to Heligmosomoides polygyrus.

Infections induced in NIH mice by irradiated (300 Gy) larvae of Heligmosomoides polygyrus effectively stimulated immunity to challenge, whereas unirradiated larvae did not. Importantly, this difference was lost by the elimination of the adult worms arising from unirradiated sensitising infections by drug treatment prior to challenge. No difference in the level of parasite-specific serum and mucosal IgG, IgG1, IgG2a, or IgA was detected between immune mice sensitised either with drug-abbreviated unirradiated or irradiated larval infections and non-immune mice receiving two superimposed unirradiated infections. An enzyme-linked immunosorbent assay (ELISA) and immunoblotting data suggested that parasite-specific IgG1 was the predominant antibody class in both serum and intestinal perfusates. IgA exhibited differences in antigen specificity between the serum and the intestine. In serum, IgA responses were directed predominantly to L4 somatic antigens, whereas at the mucosal surface they were biased towards L4 excretory/secretory (ES) antigens. No correlation was found between the intensity of the serum or mucosal antibody responses and the mean worm burdens in groups of immune or non-immune mice. Moreover, no correlation was found between levels of parasite-specific serum or mucosal IgG, IgG1, IgG2a or IgA and the loss of worms in individual mice.

The effects of gamma radiation on the development of Heligmosomoides polygyrus bakeri in mice.

The development of a Heligmosomoides polygyrus bakeri (H. polygyrus) primary infection in its definitive host was severely effected by a wide range of gamma radiation doses (10-400 Gy). Male worms were more susceptible to gamma radiation than female worms. A dose of 400 Gy prevented the development of L3 larvae to mature female worms and 200 Gy abrogated the maturation of males. At 300 Gy, a dose known to stimulate high levels of protective immunity, male worms were unable to moult to the L4 stage and females failed to develop into morphologically normal adults. An experiment to select for a radiation resistant parasite line provided data on the cumulative effects of gamma rays on successive parasite generations. Parasite fitness data demonstrated that worm development, at the level of embryogenesis, was far more sensitive to radiation damage than either post embryonic development or adult worm fecundity. The parasite line died out on the 14th generation of selection after receiving an accumulated dose of 420 Gy. It is concluded that gamma radiation profoundly alters the developmental biology of H. polygyrus in a dose-dependent manner, with maximal sensitivity exhibited during embryogenesis.