Multiple bovine FcγRIIb sub-isoforms generated by alternative splicing

Veterinary Immunology and Immunopathology 135 (2010) 43–51

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Research paper

Multiple bovine FcgRIIb sub-isoforms generated by alternative splicing Matthew A. Firth *, Kuldeep S. Chattha, Douglas C. Hodgins, Patricia E. Shewen Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1

A R T I C L E I N F O

A B S T R A C T

Article history: Received 7 August 2009 Received in revised form 21 October 2009 Accepted 22 October 2009

Receptors for the Fc portion of immunoglobulin molecules (FcR) provide an important and vital link between circulating antibody and cellular effector functions. These receptors have been well characterized in human and murine species, however few of these receptors have been investigated in livestock. FcgRII (CD32) is an FcR previously shown in mice and humans to exist in multiple isoforms, both activating (FcgRIIa, FcgRIIc) and inhibitory (FcgRIIb), on a wide variety of cells including B cells, T cells, dendritic cells, monocytes, macrophages and platelets. On B cells, FcgRIIb acts to suppress cell activation and immunoglobulin production by means of an intracellular immunoreceptor tyrosinebased inhibitory motif signaling domain. Two sub-isoforms of FcgRIIb, designated b1 and b2, distinguished by the inclusion of an additional cytoplasmic exon in the b1 form, have been demonstrated in humans and mice, whereas only one sequence corresponding to the human and mouse b2 isoform has been identified in cattle. In this study, the expression profile of FcgRIIb in bovine blood mononuclear cells was characterized by collecting blood samples from mature cattle of dairy and beef breeds, and determining their FcgRIIb mRNA expression profile by RT-PCR. Analysis revealed the presence of two uncharacterized bovine FcgRIIb transcripts in addition to the single previously published transcript. Analysis of the first unknown transcript revealed high homology with published human and murine FcgRIIb1 sequences. This transcript was present in all cell types examined, with little variation in primary sequence between individuals or among breeds. The second unknown sequence was found to be homologous to the murine FcgRIIb3 (IgGbinding protein or soluble FcgR in humans) sequence. This transcript appears to have a much more limited expression profile, which may indicate that expression varies with the cellular activation-state of the cell. These results indicate that cattle, like humans and mice, express multiple sub-isoforms of FcgRIIb. These findings add further complexity to the regulation of IgG-mediated immunity and provide new insight into the role Fc receptors play in antigen acquisition and presentation in cattle. ß 2009 Elsevier B.V. All rights reserved.

Keywords: CD32 Bovine Neonatal FcgRIIb

1. Introduction The subtleties of immune response are closely linked to signals cells receive from ligation of their surface receptors. Receptors for IgG Fc, (FcgR), are a diverse family of cellsurface receptors. At least six different FcgR (FcgRI, FcgRII, FcgRIII, FcgRIV, the bovine-specific Fcg2R, and the FcRn

* Corresponding author. Tel.: +1 519 824 4120x54468; fax: +1 519 824 5930. E-mail address: mfi[email protected] (M.A. Firth). 0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.10.029

neonatal receptor) have been identified to date, however they have been characterized in only a limited number of species (Kacskovics, 2004; Nimmerjahn and Ravetch, 2006; Qiao et al., 2006; Roopenian and Akilesh, 2007). These receptor isoforms and their respective sub-isoforms differ in binding affinities and signaling capabilities due to sequence variation and alternative splicing of mRNA transcripts. FcgRII, also known as CD32, is a low-affinity receptor for IgG that acts as an important negative regulator of B cell function (Minskoff et al., 1998; Tzeng et al., 2005; Joshi et al., 2006). FcgRII is considered to be the only FcgR expressed on B cells, and is thought to play a

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vital role in regulating cellular activation/maturation and antibody production, and in maintaining peripheral tolerance to prevent immune-mediated disease (Bolland and Ravetch, 2000; McGaha et al., 2005; Nimmerjahn and Ravetch, 2006; McGaha et al., 2008). However, expression is not limited to B cells, as FcgRII has been detected on nearly all cell types of the immune system (Brooks et al., 1989; Lynch, 2000; Su et al., 2007). Notably, expression by immature dendritic cells (DCs) has been implicated in preventing spontaneous activation and maturation (Nimmerjahn and Ravetch, 2006; McGaha et al., 2008). FcgRII expression on DCs also facilitates uptake of antibody/ antigen complexes for processing and presentation to CD4+ cells via MHC class II, and to CD8+ cells by means of cross presentation on MHC class I molecules, enhancing Ag-specific T cell responses (Yada et al., 2003; de Jong et al., 2006). FcgRII has also been detected on T cells, mast cells, monocytes, macrophages, and some epithelial and endothelial cell lineages, where the primary function appears to be antibody mediated uptake of antigen, in addition to modulation of cellular activation and maturation events (Daeron, 1995; Drechsler et al., 2002; McGaha et al., 2005). An additional layer of complexity is demonstrated by the existence of sub-isoforms of FcgRIIb, classified as b1 and b2. Achieved by variations in mRNA splicing, these subisoforms differ structurally by the addition of a short cytoplasmic exon in the b1 form, the length of which varies among species (Ravetch et al., 1986; Brooks et al., 1989; Yamashita et al., 1993). The two variants display no notable difference in their signaling capabilities, as both can undergo tyrosine phosphorylation, enhancing SHIP phosphorylation, and downregulating cellular endocytosis (Joshi et al., 2006). However, the b1 form uniquely prevents uptake of antigen/ antibody complexes via clathrin-coated pits (Amigorena et al., 1992; Miettinen et al., 1992; Minskoff et al., 1998; Joshi et al., 2006). This is attributable to an increased affinity of the b1 insertion for cytoskeletal components, resulting from the association of b1 proteins with actin molecules and membrane phospholipids (Miettinen et al., 1992; Chen et al., 1999). Since immune complexes bound to FcgRIIb1 cannot be processed through the endocytic pathway, clonotypic expansion of Ag-specific T cells is prevented due to the lack of MHC-II peptide presentation (Amigorena et al., 1992; Minskoff et al., 1998). By these means, inhibition of cellular responses can be extended beyond the cells expressing the receptor to include potential downstream effector cell populations. Less well known than the membrane-bound FcgR are the soluble forms of these receptors, first characterized in mice over 30 years ago (Fridman and Golstein, 1974; Newport-Sautes et al., 1975). Soluble FcgRIIb is generated either by the cleavage of membrane-bound proteins, or as soluble splice variants lacking the exon coding for the transmembrane (TM) region (Fridman et al., 1993; Tartour et al., 1993; Galon et al., 1997). Membrane-cleaved soluble FcR could be produced by virtually any cell expressing the full-length protein, but seem to be produced mainly by lymphocytes, at least in mice (Fridman et al., 1993). The TM negative (TM) form however, is typically produced only by cells capable of expressing FcgRIIb2, such as T cells,

macrophages and Langerhans cells, and is present in higher serum concentrations than the membrane-cleaved version (Fridman et al., 1993; Tartour et al., 1993). Soluble FcgRII have been demonstrated to regulate antibody production in an isotype-specific manner by blocking suppressor T cell functions (Simpson et al., 1996) and to affect antigen uptake and processing by epidermal Langerhans cells (Esposito-Farese et al., 1995). In many livestock species (including cattle, pigs, horses, sheep and goats) the fetus does not receive immunoglobulin by placental transfer, but relies instead on gut absorption of maternal immunoglobulin from colostrum immediately post-parturition. During the first hours of life, immunoglobulins are absorbed undigested through epithelial layers and enter the neonatal circulation (Tizard, 2008). These maternal antibodies protect the neonate from common pathogens, but unfortunately suppress neonatal active antibody responses (Glezen, 2003). This is hypothesized to be due in part to immune complexes containing antigen and maternal antibody interacting with FcgRII on neonatal antigen presenting cells. Although the specific FcgRII sub-isoform to which the immune complex binds may influence the subsequent immune response, little research has been undertaken to determine if these various splice variant sub-isoforms are present in livestock species. To obtain greater understanding of the interaction between maternal IgG and the neonatal immune system, we further characterized bovine FcgRII by examining the expression profiles of peripheral blood mononuclear cells (PBMCs) and various fluorescence-activated cell sorted (FACS) leukocyte populations by reverse-transcription and polymerase chain reaction (PCR). 2. Materials and methods 2.1. Animal subjects—sample collection BL3 cells, a bovine B cell lymphoma cell line (ATCC CRL 8037) were maintained in RPMI 1640 (Sigma, St. Louis, MO) supplemented with 7% heat-inactivated FBS, 60 mg/ ml penicillin and 50 mg/ml streptomycin. PBMCs were obtained from whole blood samples collected in acid citrate dextrose anticoagulant from mature cattle of dairy and beef breeds. Sampled breeds included Ayrshire (AYR, n = 5), Jersey (JER, n = 3), Guernsey (GRN, n = 5), Brown Swiss (BS, n = 5), Holstein (HOL, n = 2), Hereford (HER, n = 5) and Angus (ANG, n = 4). Holstein cattle were maintained by the University of Guelph at the Elora Dairy Research Station (Elora, Ontario). Cattle of other breeds of interest were maintained in commercial herds in the vicinity of the University of Guelph. All procedures involving live animals were approved by the Animal Care Committee of the University of Guelph and were in accordance with the recommendations of the Canadian Council on Animal Care. 2.2. Cell separation and RNA extraction Peripheral blood mononuclear cells were isolated from whole blood by density gradient centrifugation using

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Histopaque-1077 (Sigma) followed by two washings with Hank’s buffered saline solution. Neutrophils were isolated from the red cell fraction following centrifugation of whole blood. RBCs were removed by hypotonic lysis using sterile water, followed by addition of 3 PBS. All steps were carried out at 22 8C. RNA was isolated using the TRIzol reagent (Invitrogen, Mississauga, ON) according to the manufacturer’s instructions. 2.3. Flow cytometry Approximately 1–5  108 PBMCs were labeled with mAbs for 25 min at 4 8C. Samples were sorted by flow cytometry using FACSAria 1 (BD Sciences, Franklin Lakes, NJ) and data were analyzed for purity by FACSDiva 5.0.2 software. Sorts that resulted in cell purity greater or equal to 98% were used for RNA extractions. mAbs used in this study were MM1A-FITC (Mouse anti-bovine CD3, Veterinary Medical Research & Development (VMRD), Pullman WA); CC8-FITC (Mouse anti-bovine CD4, AbD Serotec, Raleigh, NC); CC63-PE (Mouse anti-bovine CD8, AbD Serotec); MM8 (Mouse anti-bovine CD14, VMRD); CC21 (Mouse anti-bovine CD21, AbD Serotec); X56-APC (Rat aMouse IgG1, BD Pharminogen). 2.4. PCR conditions Primers were designed to amplify the full-length FcgRII mRNA by annealing to 50 and 30 UTRs, based on a previously characterized bovine FcgRII sequence (Zhang et al., 1994). Reactions were carried out in 50 ml volumes using Platinum Taq DNA polymerase (Invitrogen, Mississauga, ON) in the presence of 2 mM Mg2+, 10 mM dNTPs, and primer concentrations of 100 mg/ml unless specified otherwise. PCR cycling conditions were 94 8C for 5 min, followed by 35 cycles of 1 m (94 8C), 30 s (60 8C), 30 s (72 8C), with an additional 10 m at 72 8C as the final extension step. FcgRII amplicons were cloned into the pCR 2.1 TOPO TA cloning vector (Invitrogen) for sequencing using an ABI Prism 3500 Capillary sequencer.

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each resultant clade, a bootstrap neighbour-joining analysis with 1000 replications was also performed.

3. Results 3.1. FcgRII transcripts in BL3 cells Gel images of PCR amplicons obtained from BL3 cells contained two bright bands of similar size, and an additional, smaller band (Fig. 1A). Sequencing of the larger amplicons revealed the presence of a single ORF in each amplicon, 951 and 894 bp in length. These ORFs were found to be identical in sequence with the exception of a 57 bp inframe insertion in ORF951. ORF894 was 99% identical with the previously published bovine FcgRIIb sequence (Zhang et al., 1994). The smallest amplicon was found to contain an ORF 774 bp in length and 99% similar to the Zhang et al. (1994) sequence, except for the absence of a 120 bp region corresponding to the predicted TM exon. A similar transcript has been described in mice, designated as the murine FcgRIIb3 variant, and is generated by alternative splicing to remove the TM exon (Tartour et al., 1993). 3.2. FcgRII transcripts in PBMCs To ensure the observed results in BL3 cells were not a result of altered expression in this laboratory lymphoma cell line, we surveyed amplicons from PBMCs obtained from individual animals of a variety of cattle breeds, using the same primers as for the BL3 cells. No major differences from the BL3 data were observed, in that all three subisoforms were detected in all individuals of all breeds examined. The only notable difference appeared to be the

2.5. Sequence analysis Sequence data were analyzed using the Geneious software package (Drummond et al., 2006). Pairwise alignments were carried out using NCBI BLAST (http:// blast.ncbi.nlm.nih.gov/Blast.cgi), multiple alignments were generated using the ClustalW standalone program (http://www.ebi.ac.uk/Tools/clustalw2/index.html) or performed manually. Detailed analysis of the published genomic sequence for the region including the CD32 locus, obtained from the Bos taurus genome (build 3.1), was carried out using the GenomeScan intron/exon prediction online software package (http://genes.mit.edu/genomescan.html). To determine the degree of sequence variation between breeds, sequences were aligned manually using Se-Al (v2.0a11 Carbon, http://tree.bio.edu.ac.uk/software/ seal/). A phylogeny was constructed using PAUP* v4.0b10 (Swofford, 2003) under the HKY85 model of nucleotide substitution as determined by Modeltest v3.7 (Posada and Crandall, 1998). To assess the levels of nodal support for

Fig. 1. Gel images of FcgRIIb amplicons. (A) Full-length amplicons obtained from BL3 cells, bovine PBMCs, and FACS sorted CD21+ cells. Note the presence of two bands of very similar size, and a faint smaller band, seen most clearly in the BL3 sample. (B) Primers were redesigned based on sequence analysis to amplify the transmembrane and cytoplasmic domains. The presence of the larger splice variants is clear in all cells examined, while the smaller variant appears clearly in only two samples. Intensity differences may indicate differences in expression levels between cell types.

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Fig. 2. (A) Exon distribution predictions were made using the Massachusetts Institute of Technology online GenomeScan software (http://genes.mit.edu/ genomescan.html), which accepts genomic sequence and a proposed amino acid sequence, and uses this information to predict intron/exon boundaries. The program identified the 57 bp exon (boxed) when either b1 or b2 protein sequence was used as input. (B) Schematic representation demonstrating exons, which make up the extracellular, transmembrane and cytoplasmic sequences. Location of primers used for splice variant screening is indicated by black boxes. These primers were designed to anneal to the flanking region used to define b1, b2 and TM splice variants.

variable inclusion of an in-frame 3 bp (GCA) insertion at the junction of cytoplasmic exons 2 and 3 in several individuals, resulting in the addition of an alanine residue at position 45 of the protein sequence (Fig. 2). This insertion was not restricted to any particular FcgRII subisoform, breed nor individual. In two individuals, an 828 bp variant of the soluble isoform was detected, which lacked the 120 bp TM exon yet contained the 57 bp hallmark exon of the b1 sub-isoform.

This provided greater resolution between bands for optimal identification of the splice variant bands by agarose gel electrophoresis. Cells were labeled as indicated in the figure and sorted by FACS. In all examined cell types, both b1 and b2 forms were visible. Differing intensities between bands were noted, but precise quantification was not possible. However, the transcript for the TM variant was faint for all cell types examined, if visible at all. 3.4. Sequence analysis of FcgRII transcripts

3.3. Detection of FcgRII transcripts in FACS sorted lymphocytes As PBMCs comprise a heterogeneous cell population, several (n = 3) animals were selected to further examine the expression profiles of individual cell populations by PCR screening. A new forward primer was designed to anneal immediately upstream of the TM region, and used in conjunction with the previous reverse primer (Fig. 1B).

GenomeScan indicated the likelihood of an additional exon not accounted for in the published FcgRIIb mRNA (Zhang et al., 1994) sequence (Fig. 2A). The length and sequence of the predicted exon matched exactly that of the 57 bp insertion noted in the previously isolated bovine sequences. BLASTp analysis of translated ORFs from the isolated transcripts identified b1 and b2 FcgRII subisoforms, based on similarity with published human b1

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Table 1 FcgRIIb sequences used in this study. Species

Isoform

ACCN

Sus scrofa

b1 b2

N/A NC_010446.1

298

Nucleotide sequence, AA seq obtained by translation of ORF

Equus caballus

b1

NW_001867418.1

323

Genomic contig—gene structure predicted by computational means, and AA sequence predicted from resulting data

b2

N/A

N/A

b1 b2

BAA02852.1 BAA02851.1 BAA02850.1

341 313 297

Rattus norvegicus

b1 b2

ABO09816.1 NP_786932.1

342 285

Mus muscularus

b1

NP_001070657.1

340

Cavia porcellus

Size (AA)

Description

Three published sequences, two representative chosen for alignments

Multiple sequence variants for each sub-isoform, two representative sequences were chosen for alignment purposes

b2

NP_034317.1

293

Macaca mulatta

b1 b2

XP_001118081.1 N/A

313 N/A

Macaca fascicularis

b1 b2

N/A AF485814_1

N/A 294

Pan troglodytes

b1 b2

XP_001153863.1 N/A

305 N/A

Sequence available only for b1 sub-isoform

Homo sapiens

b1

NP_003992.3

310

Multiple sequence variants for each sub-isoform, two representative sequences were chosen for alignment purposes.

b2

NP_001002273.1

290

Sequence available only for b1 sub-isoform

Sequence available only for the b2 sub-isoform

Accession numbers (ACCN) for protein sequences are given where available. For organisms where no protein sequence was published, an amino acid (AA) sequence was generated by computational analysis. Where multiple sequence variants existed, single representative sequences were chosen for comparison, as indicated. (N/A indicates sequence data not available.)

(59.2%) and b2 (57.6%) sequences. These sequences were then compared to current published FcgRIIb protein sequences for a variety of species summarized in Table 1, and the alignments presented in Fig. 3. Pairwise BLASTp analyses were run to generate a similarity matrix for current published FcgRIIb sequences (Table 2). Due to the lack of published FcgRIIb sequences, many species are represented by only a single sequence. For those species where multiple transcript variants exist for each subisoform (namely human and murine), single representative sub-isoform variants were selected. Phylogenic analysis of the collected b1, b2 and b3 sequences showed no clustering by breed, suggesting a lack of breed-specific

differences in FcgRIIb (Fig. 4). Differences in primary sequence between individuals are therefore more likely to be due to animal-specific single nucleotide polymorphisms (SNPs), however data from more animals per breed and from additional breeds and species of cattle (such as Bos indicus) would be required to resolve this question. 4. Discussion and conclusions The identification of additional FcgRIIb sub-isoforms in livestock species is of great interest to the field of comparative immunology. Knowledge that the FcgR receptor family in cattle includes not a single, but multiple

Table 2 Pairwise similarity matrix comparing protein sequences for FcgRIIb across all examined species. ClustalW scores are given for b1 comparisons (upper right) and for b2 comparisons (lower left).

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Fig. 3. Multispecies alignment of FcgRIIb1 and b2 sequences. Protein sequences for FcgRIIb sequences were collected from NCBI and aligned by ClustalW. Green bars in the identity matrix indicate regions of high identity among all examined sequences, while regions of low identity are indicated in red. Yellow indicates moderate identity. The red arrow shows the minimum b1 insertion motif required to affect function. Note that several species, mostly rodent, possess additional amino acid residues in this region. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

forms of FcgRIIb has the ability to shed new light on the understanding of important immunological processes occurring at the cellular level. A crucial event in the life of newborns of all domesticated livestock species is the ingestion and uptake of maternal IgG in colostrum. Maternal antibodies absorbed from colostrum protect the neonate from many infections while the immune system matures to an adequate functional level. This protection can be achieved by opsonization of bacteria, virus neutralization, or neutralization of toxins (Tizard, 2008). However, following vaccination or the acquisition of an infection by the young animal, immunoglobulins received passively via colostrum cause the formation of immune complexes. These IgG

containing immune complexes have potential to bind FcgRII on neonatal cells, exerting influence over the subsequent immune response. Previous research has demonstrated that animals of a very young age express surface FcgRII on several cell types (Chattha et al., 2009). Neonatal animals are also capable of mounting immune responses, as demonstrated by early life vaccination studies (Siegrist et al., 1998; Hodgins and Shewen, 2000; Dadaglio et al., 2002). Whether this is due to classical uptake and presentation of antigen by B cells via FcgRIIb2 and MHC-II, respectively, or by display of Ab/Ag complexes on DC cell surfaces via FcgRIIb1 is unknown (Bergtold et al., 2005). While the inhibitory outcome has been well documented in vitro with respect to human and murine

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Fig. 4. Tree diagram of aligned FcgRIIb1, b2 and b3 sequences, generated using PAUP* v4.0b10. Branches are colored to indicate members of the same breed (Holstein—HOL, Jersey—JER, Brown Swiss—BS, Guernsey—GRN, Ayrshire—AYR, Hereford—HER and Angus—ANG). No bootstrap values are reported, as all values were below 60% support. The topology of the chart and low bootstrap values indicate that there are no significant differences among breeds with respect to FcgRIIb sequences. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

B cells, little to no research has been undertaken with respect to neonates in general or calves in particular. We recently examined RNA isolated from PBMCs from calves of a variety of ages, ranging from 2 days to 8 months, and were able to detect transcript for both the b1 and b2 subisoforms, however definitive determinations of the ratio of b1:b2 expression could not be made (data not shown).

Knowledge of the expression profiles of the b1 and b2 proteins in various cells and tissues would undoubtedly provide valuable insight into neonatal cellular immune responses. Notably, our analysis of the FcgRIIb sequences obtained from mature individuals of all sampled breeds of cattle indicates the potential for presence of all three FcgRIIb

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sub-isoforms, with a low degree of individual variance (>98% similar). Therefore, it is reasonable to conclude that FcgRIIb variants are widely distributed in B. taurus cattle, and differ very little in their primary sequences between B. taurus breeds. Protein sequences for FcgRIIb were compared across several species to identify conserved regions. Some difficulty was encountered in identifying the correct sub-isoforms to be compared, as the receptor naming conventions have been based on order of discovery and not sequence homology. For example, the sequence identified as FcgRIIb1 in the guinea pig does not contain the b1-exon insert, while the sequences identified as b2 and b3 do (Yamashita et al., 1993). The guinea pig b3 sub-isoform also contains the TM sequence, unlike its murine counterpart. Similarly, the sequence for bovine FcgRII deposited by Zhang et al. (1994) is identified as the b1 sub-isoform, however it lacks the 57 bp b1 insertion. This is a common problem in immunology, and often the fact two proteins share a name between species does not imply equivalent function. This is of particular note, considering the high homology found upon comparison of the cytoplasmic region of FcgRIIb (containing the b1-hallmark sequences) (Fig. 3). While the length of the b1-insert varied among the species examined (ranging from 15 to 46 amino acid residues), the high degree of cross-species similarity suggests that sub-isoforms should be given equivalent names. Longer b1 insertions were found in rodents such as mice and guinea pigs (Yamashita et al., 1993; Latour et al., 1996), while shorter sequences were noted in primates (including humans) and livestock species. The motif responsible for preventing endocytosis of b1 proteins has previously been mapped to the N-terminal region of this insert using several deletion mutants (Miettinen et al., 1992). Strikingly, this region appears conserved across all examined species (Fig. 3), and is likely to represent the minimum motif required to abrogate endocytosis. The presence in rodents of additional residues has yet to be explained. We have attempted to maintain a naming convention for our newly discovered isoforms consistent with published human and murine sequences, and recommend the re-classification of the existing Zhang sequence as the b2 sub-isoform. The identification of an alternatively spliced form of bovine FcgRIIb lacking the TM region is of particular interest, as this is the first time this form has been described in a species other than mouse or human. While little has been published regarding the importance of soluble FcRs, in vitro studies have shown they may modify several immunological processes. Isotype selection, B cell activation, and modulation of receptormediated endocytosis have been shown to be influenced by soluble FcR (Esposito-Farese et al., 1995; Simpson et al., 1996; Galon et al., 1997). While the detection of this splice variant is of great interest, elucidation of the precise biological function is beyond the scope of the current research. This topic is certainly a key area for future examination. Results obtained from transcription profiling of the FACS sorted PBMCs have interesting implications, as it appeared that the b1 and b2 sub-isoforms display

differences in their ratio of expression among cell types. In fact, these sub-isoforms seem to be limited in their expression, as indicated by the absence or faint indication of bands obtained from specific cell populations (Fig. 1B). The presence of bands for both b1 and b2 sub-isoforms in the CD21+ cell population is of particular interest, as previous research has indicated that while murine B cells preferentially express the b1 form, human B cells produce transcript for both sub-isoforms, as was found in cattle (Amigorena et al., 1989; Ravetch and Kinet, 1991; Cassel et al., 1993). A potential limitation of this study is the lack of functional data relating to the sub-isoforms described above. Since current anti-bovine FcgRII mAbs (anti-CD32) are specific to the extracellular domain, it is not possible to discern which sub-isoform is being expressed at the cell surface. This is because the two sub-isoforms differ in the cytoplasmic region, immediately adjacent to the plasma membrane. mAb specific for this epitope may be needed in order to examine this topic in greater detail. However, the knowledge that these sub-isoforms are present in cattle should encourage future studies to determine their function. Currently, our laboratory is examining the relative mRNA expression of the various sub-isoforms in different cell lineages, and variation of expression with age. In conclusion, this study has demonstrated the expression of three sub-isoforms of FcgRIIb, two cell-associated and one soluble, that were previously unknown in cattle. In light of this knowledge, previous predictions of cell activity based on use of the existing anti-CD32 mAb, will need to be re-examined. Further, since these sub-isoforms likely implicate diverse signaling pathways, previous models of antibody mediated regulation of B cell activation, particularly in neonates, should be re-assessed. Conflict of interest None of the authors of this paper are subject to any conflict of interest with respect to the research reported in this document. Acknowledgements This work was financially supported by the Natural Sciences and Engineering Research Council (NSERC), the Canadian Cattlemen’s Association (Beef Cattle Research Council), the Dairy Farmers of Canada, the Ontario Cattlemen’s Association (Agricultural Adaptation Council), the Alberta Beef Producers, the Alberta Livestock and Meat Agency, and the Ontario Ministry of Agriculture, Food and Rural Affairs. M. Firth is the 2006 recipient of the Brock Doctoral Scholarship at the University of Guelph. We would like to thank Cadhla Firth for her assistance and consultation on the phylogenic analysis, B.A. McBey for providing technical assistance with FACS and animal sampling, and Angela Holiss and the University of Guelph Genomics Facility staff for DNA sequencing. The assistance and cooperation of the staff of the Ponsonby and Elora Dairy Research Stations, and private herd owners were greatly appreciated.

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