H. Craig Morton

The B-cell system of human mucosae and exocrine glands.

Summary: The mucosae and exocrine glands harbour the largest activated B‐cell system of the body, amounting to some 80–90% of all immunoglobulins (Ig)‐producing cells. The major product of these immunocytes is polymeric (p)IgA (mainly dimers) with associated J chain. Both pIgA and pentameric IgM contain a binding site for the polymeric Ig receptor (pIgR), or secretory component (SC), which is a requirement for their active external transport through secretory epithelia. The pIgR/SC binding site depends on covalent incorporation of the J chain into the quaternary structure of the polymers when they are produced by the local immunocytes. This important differentiation characteristic appears to be sufficient functional justification for the J chain to be expressed also by most B cells terminating at secretory effector sites with IgD or IgG production; they probably represent a ‘spin‐off’ from sequential downstream CH switching on its way to pIgA expression, thus apparently reflecting a maturational stage of effector B‐cell clones compatible with homing to these sites. Observations in IgA‐deficient individuals suggest that the magnitude of this homing is fairly well maintained even when the differentiation pathway to IgA is blocked. Certain microenvironmental elements such as specific cytokines and dendritic cells appear to be required for induction of IgA synthesis, but it remains virtually unknown why this isotype normally is such a dominating product of local immunocytes and why they have such a high level of J chain expression. Also, despite the recent identification of some important requirements in terms of adhesion molecules (e.g. integrin α4β7 and MAdCAM‐1) that explain the “gut‐seeking” properties of enterically induced B cells, the origin of regionalized homing of B cells to secretory effector sites outside the gut remains elusive. Moreover, little is known about immune regulation underlying the striking disparity of both the class (IgD, IgM) and subclass (IgA1, IgA2, IgGI, IgG2) production patterns shown by local iinmttnocytes in various regions of the body, although the topical microbiota and other environmental stimuli might be important. Rational design of local vaccines will depend on better knowledge of both inductive and migratory properties of human mucosal B cells.

FUNCTIONAL ASSOCIATION BETWEEN THE HUMAN MYELOID IMMUNOGLOBULIN A FC RECEPTOR (CD89) AND FCR GAMMA CHAIN : MOLECULAR BASIS FOR CD89/FCR GAMMA CHAIN ASSOCIATION

FcR γ chain has previously been shown to interact with the TCR-CD3 complex, the IgE Fc receptor I (FcεRI), and the class I and IIIA IgG receptors (FcγRI and FcγRIIIa). Here, we demonstrate that the Fc receptor γchain associates with FcαR in transfected IIA1.6 B lymphocytes. FcαR could be expressed at the surface of IIA1.6 B cells by itself, but was devoid of signaling capacity. Upon co-expression of FcR γchain, a physical interaction with FcαR could be demonstrated. This association proved crucial for the triggering of both proximal (intracellular calcium increase and tyrosine phosphorylation), as well as distal (IL-2 release), signal transduction responses. We next tested the hypothesis that a positively charged arginine residue (Arg209) within the transmembrane domain of FcαR promotes association with FcR γchain. We therefore constructed FcαR molecules where Arg209 was mutated to either a positively charged histidine, a negatively charged aspartic acid, or an uncharged leucine. A functional association between FcαR and FcR γchain was observed only with a positively charged residue (Arg209 or His209) present within the FcαR transmembrane domain. These data show that transmembrane signal transduction by the FcαR is mediated via FcR γchain, and that FcαR requires a positively charged residue within the transmembrane domain to promote functional association.

From B to A the mucosal way.

Generation of intestinal secretory IgA depends on antigen induction of B cells in organized GALT. A recent paper in Nature reports that in mice the lamina propria provides signals that direct mucosal B cells to undergo Cα class switching and as a basis for SIgA production.

M cell pockets of human Peyer's patches are specialized extensions of germinal centers

M cells in follicle‐associated epithelium of Peyers patches (PP) mediate antigen entrance into the underlying lymphoid tissue. To investigate the functional potential of B cells in this unique microcompartment, the expression of co‐stimulatory molecules necessary for B‐T cell interaction was examined in histologically normal human PP by three‐color immunohistochemistry. In the M cell areas, CD80 / CD86 expression was much more frequent on memory (sIgD–CD20+) B cells than on naive (sIgD+CD20+) B cells. M cell areas identified by such co‐expression of CD20 and CD80 / CD86 were always spatially related to germinal centers (GC). Contrary to the GC B cell phenotype (sIgD–CD20+CD80 / 86hiCD10+Bcl‐2–), however, M cell‐associated B cells with a high level of CD80 / CD86 were CD20loCD10–Bcl‐2+, and adjacent memory T cells (CD3+CD45R0+) often expressed CD40L (CD154). Autologous peripheral blood B‐T cell cocultures with purified protein derivative as antigen showed that the sIgD–CD80 / CD86hiCD20lo phenotype could indeed be generated during cognate B‐T interactions, concurrent with CD40L up‐regulation on memory T cells. Thus, this M cell‐associated phenotype might result from B‐T cell interactions in the course of antigen presentation by memory B cells, with subsequent CD40 engagement by CD40L‐expressing cognate memory T cells. We propose that this M cell‐associated event contributes to memory B cell survival and diversification of intestinal immunity, representing a specialized limb of GC function.

Regulation of switching and production of IgA in human B cells in donors with duplicated α1 genes

IgA is the predominant immunoglobulin class synthesized in humans and can be subdivided into two subclasses, IgA1 and IgA2, each encoded by a separate gene and differentially expressed depending on age and anatomical localization of the producing cells. Duplication of the α1 gene is frequently observed in selected populations. As this duplication may serve to enhance IgA‐mediated immunity, we determined its effect on switching and production of IgA in human B cells. We developed a nested PCR strategy, involving sequencing the switch (S) α2 region, the only human S region not sequenced to date, to assess the proportion of cells switching to IgA1 and IgA2 in vivo. Our results show that there is no difference in the serum and salivary levels of IgA1 and IgA or rate of switching to IgA1 and IgA between normal donors and individuals carrying α1 gene duplications, suggesting involvement of a regulatory step in the production of IgA.

Cloning and characterization of an immunoglobulin A Fc receptor from cattle

Here, we describe the cloning, sequencing and characterization of an immunoglobulin A (IgA) Fc receptor from cattle (bFcαR). By screening a translated EST database with the protein sequence of the human IgA Fc receptor (CD89) we identified a putative bovine homologue. Subsequent polymerase chain reaction (PCR) amplification confirmed that the identified full‐length cDNA was expressed in bovine cells. COS‐1 cells transfected with a plasmid containing the cloned cDNA bound to beads coated with either bovine or human IgA, but not to beads coated with bovine IgG2 or human IgG. The bFcαR cDNA is 873 nucleotides long and is predicted to encode a 269 amino‐acid transmembrane glycoprotein composed of two immunoglobulin‐like extracellular domains, a transmembrane region and a short cytoplasmic tail devoid of known signalling motifs. Genetically, bFcαR is more closely related to CD89, bFcγ2R, NKp46, and the KIR and LILR gene families than to other FcRs. Moreover, the bFcαR gene maps to the bovine leucocyte receptor complex on chromosome 18. Identification of the bFcαR will aid in the understanding of IgA–FcαR interactions, and may facilitate the isolation of FcαR from other species.

Cloning and characterization of equine CD89 and identification of the CD89 gene in chimpanzees and rhesus macaques

Immunoglobulin A (IgA) is the major antibody class present in external secretions of mammals. At the vulnerable mucosal surfaces, IgA provides a crucial first‐line defence by neutralizing pathogens. Primates also have a substantial level of IgA in serum and although not well understood, the biological role of this IgA depends, at least partly, on its ability to interact with specific receptors (FcαRs) on the surface of leucocytes. The human FcαR, CD89, was the first IgA Fc receptor to be identified and binding of IgA‐coated particles to CD89 triggers numerous cellular effector functions, including phagocytosis, antibody‐dependent cellular cytotoxicity, and release of inflammatory mediators, all of which may play an important role in both systemic and mucosal immunity. For many years humans were the only species known to express CD89, however, it has recently been cloned from cows and rats. Here, we describe the identification of the CD89 gene in three additional species: horses, chimpanzees, and Rhesus macaques. Equine CD89 was identified at the cDNA level, whereas the chimpanzee and Rhesus macaque genes were identified from the available draft genomic sequence. Interestingly, when compared with humans and other primates, horses, cows and rats have a relatively low concentration of serum IgA, so the role of CD89 in these species is of particular interest. The identification and characterization of CD89 in different species will contribute to a greater understanding of the biological role of IgA and CD89 in mucosal and systemic immunity throughout evolution.

Identification of residues within the EC1 domain of bovine Fcγ2R essential for binding bovine IgG2

Neutrophils and monocytes in cattle express a novel class of immunoglobulin Fc receptor, specific for bovine IgG2 (bIgG2), termed bFcγ2R. In cows, the ability of neutrophils to kill immunoglobulin-opsonized microorganisms appears to depend largely on this subclass, whose interaction with bFcγ2R initiates the killing process. bFcγ2R is a transmembrane glycoprotein consisting of two extracellular immunoglobulin-like domains, followed by a 19-amino acid membrane-spanning region and a short cytoplasmic tail. Although related to other mammalian FcγRs, bFcγ2R belongs to a novel gene family that includes the human killer cell inhibitory receptor and FcαRI (CD89) proteins. We have shown previously (Morton, H. C., van Zandbergen, G., van Kooten, C., Howard, C. J., van de Winkel, J. G., and Brandtzaeg, P. (1999) J. Exp. Med. 189, 1715–1722) that like these proteins (and unlike other FcγRs), bFcγ2R binds bIgG2 via the membrane-distal extracellular domain 1 (EC1). In this present study, we introduced mutations into the predicted loop regions of the EC1 domain and assayed the resulting bFcγ2R mutants for their ability to bind bIgG2. Our results indicated that the bIgG2 binding site lies within the predicted F–G loop region of the EC1 domain. Furthermore, single amino acid mutational analysis of this region identified Phe-82 and Trp-87 as being critical for bIgG2 binding.

Cloning and sequencing of a cDNA encoding the bovine FcR γ chain

Abstract Phagocytic cells of the immune system express specific receptors for the Fc region of immunoglobulins (FcRs). In humans, most FcRs for IgG (FcγR), IgA (FcαR) and IgE (FceR) consist of an immunoglobulin (Ig) -binding subunit associated with a specialized signaling molecule, the FcR γ chain. The FcR γ chain is crucial for the transmission of intracellular signals following receptor ligation. In cattle, however, although four distinct complimentary DNAs (cDNAs) encoding IgG-binding subunits have been described (corresponding to bovine FcγRI, FcγRII, FcγRIII, and Fcγ2R), virtually, nothing is known about signal transduction via bovine FcRs. Therefore, in this study, a cDNA encoding the bovine FcR γ chain was cloned. The cDNA is 258 base pairs long and encodes a protein of 85 amino-acids. The mature protein shows high homology with the FcR γ chains from several other species. Interestingly, the cytoplasmic domain of the bovine FcR γ chain is one amino-acid shorter than those previously described. Cloning of a cDNA encoding, the bovine FcR γ chain will allow for a better understanding of signal transduction processes triggered by bovine FcRs.

CHAPTER 15 – Characteristics of Mucosal B Cells with Emphasis on the Human Secretory Immune System

This chapter deals with the mechanisms involved in the differentiation of mucosal B cells and signals directing their preferential homing to secretory effector sites. Although the focus is on human mucosal tissues, fundamental mechanistic information has to be extrapolated from animal experiments. The human mucosal B-cell system responds to an infection with local immunoglobulin A (IgA) and immunoglobulin M (IgM) production. Secretory immunity depends on an intimate cooperation between mucosal B cells and exocrine epithelia. The intestinal immunocytes are largely derived from B cells initially induced in gut associated lymphoid tissue (GALT). Although the B-cell migration to the intestinal lamina propria or glandular stroma is guided by the well-defined adhesion molecules, chemokines and chemokine receptors, a better definition of chemotactic stimuli determining homing mechanisms in different segments of the gut is required. In addition to homing molecules, the retention and accumulation of B cells extravasated at secretory effector sites is influenced by antigen-driven local proliferation and differentiation. However, the role of cognate T cells, MHC class II-expressing antigen-presenting cells (APCs), and epithelial cells in providing the necessary stimulatory signals remains poorly defined. It is also important to point out that the clinical observations in immunodeficient patients have shown that secretory antibodies (SIgA and SIgM), and IgG antibodies are not the only important components of the mucosal immune system. It is revealed that innate defense mechanisms are much more crucial and complex than previously believed. The cooperation between innate and adaptive immunity must be further explored to understand how the homeostasis of mucous membranes is maintained.