Preparation of the porcine secretory component and a monoclonal antibody against this protein.

Secretory component (SC) is a component of secretory IgA that is designated sIgA to distinguish it from IgA. The monoclonal antibody (MAb) against SC has been shown to be an excellent tool for the detection of the level of sIgA and for the evaluation of the efficacy of mucosal immunity. To prepare a monoclonal antibody against porcine SC, a recombinant porcine SC was expressed and purified. To develop this recombinant SC, the gene encoding the porcine SC was ligated into the pCold I vector. The recombinant vector was then transformed into Escherichia coli BL 21 (DE3), and gene expression was successfully induced by isopropyl-ß-D-thiogalactoside (IPTG). After affinity purification with Ni-NTA resin and gel recovery, the recombinant SC protein was used to immunize BALB/c mice. Finally, three hybridoma cell lines showing specific recognitions of both recombinant SC and native SC were used as stable secretors of MAbs against porcine SC and were confirmed to have no reaction to porcine IgA or IgG. The successful preparations of recombinant SC protein and MAbs provide valuable materials that can be used in the mucosal infection diagnosis for porcine disease and mucosal immune evaluation for porcine vaccine, respectively.

Human plasma-derived polymeric IgA and IgM antibodies associate with secretory component to yield biologically active secretory-like antibodies.

Immunotherapy with monoclonal and polyclonal immunoglobulin is successfully applied to improve many clinical conditions, including infection, autoimmune diseases, or immunodeficiency. Most immunoglobulin products, recombinant or plasma-derived, are based on IgG antibodies, whereas to date, the use of IgA for therapeutic application has remained anecdotal. In particular, purification or production of large quantities of secretory IgA (SIgA) for potential mucosal application has not been achieved. In this work, we sought to investigate whether polymeric IgA (pIgA) recovered from human plasma is able to associate with secretory component (SC) to generate SIgA-like molecules. We found that ∼15% of plasma pIgA carried J chain and displayed selective SC binding capacity either in a mixture with monomeric IgA (mIgA) or after purification. The recombinant SC associated covalently in a 1:1 stoichiometry with pIgA and with similar efficacy as colostrum-derived SC. In comparison with pIgA, the association with SC delayed degradation of SIgA by intestinal proteases. Similar results were obtained with plasma-derived IgM. In vitro, plasma-derived IgA and SIgA neutralized Shigella flexneri used as a model pathogen, resulting in a delay of bacteria-induced damage targeted to polarized Caco-2 cell monolayers. The sum of these novel data demonstrates that association of plasma-derived IgA or IgM with recombinant/colostrum-derived SC is feasible and yields SIgA- and SIgM-like molecules with similar biochemical and functional characteristics as mucosa-derived immunoglobulins.

Mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor.

The 17-juxtamembrane cytoplasmic residues of the polymeric immunoglobulin receptor contain an autonomous basolateral targeting signal that does not mediate rapid endocytosis (Casanova, J. E., G. Apodaca, and K. E. Mostov. Cell. 66:65-75). Alanine-scanning mutagenesis identifies three residues in this region, His656, Arg657, and Val660, that are most essential for basolateral sorting and two residues, Arg655 and Tyr668, that play a lesser role in this process. Progressive truncations suggested that Ser664 and Ile665 might also play a role in basolateral sorting. However, mutation of these residues to Ala or internal deletions of these residues did not affect basolateral sorting, indicating that these residues are probably not required for basolateral sorting. Two-dimensional NMR spectroscopy of a peptide corresponding to the 17-mer signal indicates that the sequence Arg658-Asn-Val-Asp661 has a propensity to adopt a beta-turn in solution. Residues COOH-terminal to the beta-turn (Arg662 to Arg669) seem to take up a nascent helix structure in solution. Substitution of Val660 with Ala destabilizes the turn, while mutation of Arg657 to Ala does not appear to affect the turn structure. Neither mutation detectably altered the stability of the nascent helix in the COOH-terminal portion of the peptide.

Polarized transport of the polymeric immunoglobulin receptor in transfected rabbit mammary epithelial cells.

A cDNA for the rabbit low Mr polymeric immunoglobulin (poly-Ig) receptor was expressed in an immortalized rabbit mammary cell line. The intracellular routing of the receptor and its cell surface expression was analyzed in stably transfected cells grown on permeable supports. Initially the cells formed a monolayer with no transmural electrical resistance. All monolayer cells expressed the poly-Ig receptor and cytokeratin 7 filaments characteristic of luminal mammary cells but absent in myoepithelial cells. Within 7 d in culture, the cells underwent cytodifferentiation and formed a bilayer with a transepithelial electrical resistance of approximately 500 omega x cm2. Upper layer cells formed tight junctions with adjacent cells and gap junctions with basal cells. Expression of the poly-Ig receptor and cytokeratin 7 was restricted to the cells from the upper layer. The kinetics of receptor biosynthesis and processing was similar to that reported for rabbit mammary gland and rat liver. The receptor was cleaved at the apical cell surface and release of secretory component into the apical medium occurred with a half-time of approximately 2 h. Selective cell surface trypsinization combined with pulse-chase experiments served to determine at which cell surface domain newly synthesized receptor appeared first. The receptor was digested with a half-time of approximately 60 min with trypsin present in the basolateral medium and 90 min with apical trypsin. These data are consistent with selective targeting of newly synthesized receptor to the basolateral surface. The results indicate that transcytosis of the receptor from basolateral to apical membrane in the presence or the absence of its ligand requires approximately 30 min. Cleavage of the receptor by endogenous protease is not concomitant with its appearance at the apical surface, but requires additional time, thus explaining the presence of intact receptor on the apical membrane.

Phosphorylation of the polymeric immunoglobulin receptor required for its efficient transcytosis.

The endosomal compartment of polarized epithelial cells is a major crossroads for membrane traffic. Proteins entering this compartment from the cell surface are sorted for transport to one of several destinations: recycling to the original cell surface, targeting to lysosomes for degradation, or transcytosis to the opposite surface. The polymeric immunoglobulin receptor (pIgR), which is normally transcytosed from the basolateral to the apical surface, was used as a model to dissect the signals that mediate this sorting event. When exogenous receptor was expressed in Madin-Darby Canine Kidney (MDCK) cells, it was shown that phosphorylation of pIgR at the serine residue at position 664 is required for efficient transcytosis. Replacement of this serine with alanine generated a receptor that is transcytosed only slowly, and appears to be recycled. Conversely, substitution with aspartic acid (which mimics the negative charge of the phosphate group) results in rapid transcytosis. It was concluded that phosphorylation is the signal that directs the pIgR from the endosome into the transcytotic pathway.

Smoking and serum CA19-9 levels according to Lewis and secretor genotypes.

CA19-9, a marker for cancers of biliary tract, pancreas and colorectum, is not synthesized in those with no enzyme activity genotype (le/le) of Lewis (Le) gene. No enzyme activity genotype (se/se) of secretor (Se) gene is known to have an association with high serum CA19-9 levels. There are also variations in serum CA19-9 levels independent of the genotypes. This study aimed to examine the associations of serum CA19-9 levels with smoking, alcohol drinking and body mass index (BMI; kg/m(2)), after the adjustments of Le and Se genotypes. Subjects were 486 health check-up examinees (158 males and 328 females) aged from 39 to 90 years in Hokkaido, Japan. Genotyping was conducted for 3 polymorphisms; Le T59G (59T for Le allele and 59G for le allele), Se A385T (385A for Se allele and 385T for sej allele), and Se pseudogene (se5 allele). The genotypes of Le and Se were deterministic factors of serum CA19-9. Those with Le/Le & se/se had the highest mean, while CA19-9 was not detected or very low in those with le/le. Although no associations were observed with alcohol drinking and BMI, a significant association was observed with smoking. Among those with Le/Le, the geometric mean of CA19-9 was significantly lower for current smokers than for noncurrent smokers (p = 0.011 in 4-way ANOVA with age, sex and Se genotype). When hemoglobin A1c was further adjusted, the association became stronger (p = 0.0027). In addition to polymorphic variations, some components of cigarette smoke may influence the production or destruction of CA19-9.

Local production of secretory IgA in the eye-associated lymphoid tissue (EALT) of the normal human ocular surface.

PURPOSE: Secretory IgA (SIgA) is a critical local defense mechanism of mucosal immunity. Although the conjunctiva, as part of the ocular surface, has a mucosa-associated lymphoid tissue, the production of SIgA by local plasma cells and its transport is unequivocally accepted to occur only in the upstream lacrimal gland (LG). The molecular components were therefore investigated by immunohistochemistry (IHC) and their local production verified by RT-PCR. METHODS: Tissues from 18 conjunctivas and 9 LGs of human donor eyes with normal ocular surfaces were analyzed by histology and IHC. Different zones of 12 further conjunctivas and LG tissues were analyzed by RT-PCR for the presence of the respective mRNA. RESULTS: Plasma cells were present in the diffuse lymphoid tissue of all investigated specimens and showed an intense immunoreactivity for IgA. This immunoreactivity was absent when the antiserum was preadsorbed with the protein. The luminal epithelium, with the exception of goblet and basal cells, was strongly positive for the epithelial transporter molecule secretory component (SC) in the conjunctiva and interconnecting excretory duct similar to the LG. PCR products for IgA, the monomeric IgA-joining molecule (J-chain) and SC were regularly found in all conjunctival zones and in the LG in gel electrophoresis and were sequenced. CONCLUSIONS: The local production of SIgA is for the first time verified by RT-PCR in the human conjunctiva and in the LG. This finding points to an active role of the conjunctiva in secretory immune protection of the ocular surface and supports the presence and importance of EALT at the normal ocular surface.

Characterization of the human secretory component gene promoter.

Secretory Component (SC) is a receptor molecule implicated in the transepithelial transport of polymeric immunoglobulins. We have cloned and characterized the first exon, part of the first intron and 3500 bp of the upstream region of the gene and determined the transcription initiation region. A GC rich region immediately upstream of the transcription start region is interrupted by a potential TATA-box (TTTAA) at position -28. Promoter activity was demonstrated in transient transfection experiments in HepG2 and HeLa cells. The smallest fragment still showing transcriptional activity contains 48 bp of SC promoter. A number of putative recognition sites for transcription factors possibly involved in the regulation of SC transcription by steroids, peptide hormones and cytokines were found in the upstream region.

Relationship between the secretor status and the expression of ABH blood group antigenic determinants in human intestinal brush-border membrane hydrolases.

At least six hydrolases of the human intestinal brush-border membrane bear ABH blood group antigenic determinants related to the erythrocyte phenotype: the intestinal glycoproteins of blood group A and B subjects express A or B determinants, respectively, while blood group O subjects express the H determinant identified with Ulex europaeus lectin I. These expressions are under the control of the secretor gene: ABH antigens were not detected in the hydrolases of non-secretor subjects.

Androgen specificity of a response unit upstream of the human secretory component gene is mediated by differential receptor binding to an essential androgen response element.

The expression of secretory component (SC), the epithelial receptor for poly-immunoglobulins, is regulated in a highly tissue-specific manner. In several tissues, e.g. lacrimal gland and prostate, SC synthesis is enhanced by androgens at the transcriptional level. In this study, we describe the presence of an androgen response unit, located 3.3 kb upstream of the sc transcription initiation site and containing several 5'-TGTTCT-3'-like motifs. Although each of these elements is implicated in the enhancer function, one element, the ARE1.2 motif, is found to be the main interaction site for the androgen receptor as demonstrated in in vitro binding assays as well as in transient transfection assays. A high-affinity binding site for nuclear factor I, adjacent to this ARE, is also involved in the correct functioning of the sc upstream enhancer. The ARE1.2 motif consists of an imperfect direct repeat of two core binding elements with a three-nucleotide spacer and therefore constitutes a nonconventional ARE. We demonstrate that this element displays selectivity for the androgen receptor as opposed to glucocorticoid receptor both in in vitro binding assays and in transfection experiments. Mutational analysis suggests that the direct nature of the half-site repeat is responsible for this selectivity. We have thus determined a complex and androgen-specific response unit in the far upstream region of the human SC gene, which we believe to be involved in its androgen responsiveness in epithelial cells of different organs such as prostate and lacrimal gland. We were also able to demonstrate that the primary sequence of a single nonconventional ARE motif within the enhancer is responsible for its androgen specificity.

Expression and purification of biologically active domain I of the human polymeric immunoglobulin receptor.

Previous studies using proteolytic fragments and synthetic peptides have indicated that domain I of human polymeric immunoglobulin receptor (PIgR) is necessary for ligand binding. The expression in E. coli, and subsequent IgM-affinity purification of domain I of human PIgR is described. The recombinant domain I protein (rDI) was similar in structure to native SC domain I in that it bound specifically to MAb 6G11, an antibody which recognizes a critical portion of the PIg binding site in domain I. The biological activity of rDI was indicated by high affinity binding to PIgA (Kd = 1.6 x 10(-7) M) and IgM (Kd = 5.1 x 10(-7) M). Domain I of human SC is therefore sufficient for binding to PIg.

Interferon-gamma induces polymeric immunoglobulin receptor mRNA in human intestinal epithelial cells by a protein synthesis dependent mechanism.

Transport of secretory IgA into external fluids is mediated by the polymeric immunoglobulin receptor (pIgR) on the surface of mucosal epithelial cells. We studied the mechanism by which interferon-gamma (IFN-gamma) induces pIgR expression in HT-29.74 cells, a subclone of the HT-29 cell line selected for high concns of pIgR. Here we report the isolation of genomic DNA and cDNA clones encoding human pIgR and development of a sensitive ribonuclease protection assay for pIgR mRNA. This assay was used to determine if induction of pIgR by IFN-gamma is mediated by accumulation of pIgR mRNA. After an initial lag of 12 hr, pIgR mRNA increased seven-fold in response to IFN-gamma, reaching a plateau at 24 hr. Concentrations of pIgR protein also increased seven-fold, but the increase was delayed until 48 hr following stimulation with IFN-gamma. Cycloheximide treatment abolished the IFN-gamma induced increase in pIgR mRNA, indicating that induction of pIgR mRNA by IFN-gamma requires de novo protein synthesis. These results suggest that induction of pIgR expression by IFN-gamma involves an increase in steady-state concns of pIgR mRNA via a protein synthesis dependent mechanism.

Highly inducible synthesis of heterologous proteins in epithelial cells carrying a glucocorticoid-responsive vector.

A glucocorticoid-responsive vector is described which allows for the highly inducible expression of complementary DNAs (cDNAs) in stably transfected mammalian cell lines. This vector, pLK-neo, composed of a variant mouse mammary tumor virus long terminal repeat promoter, containing a hormone regulatory element, a Geneticin resistance-encoding gene in a simian virus 40 transcription unit, and a polylinker insertion site for heterologous cDNAs, was used to express the polymeric immunoglobulin (poly-Ig) receptor and the thymocyte marker, Thy-1, in Madin-Darby canine kidney (MDCK) cells and in murine fibroblast L cells. A high level of poly-Ig receptor or Thy-1 mRNA accumulation was observed in MDCK cells in response to dexamethasone with a parallel ten- to 200-fold increase in protein synthesis depending on the recombinant protein and the transfected cell clone.

Intracellular targetting signals of polymeric immunoglobulin receptors are highly conserved between species.

A rat liver cDNA library, constructed in the plasmid expression vector pUEX, was immunoscreened using a rabbit polyclonal antiserum raised against rat liver Golgi membrane proteins. A sub-set of isolated clones were shown to encode the rat polymeric immunoglobulin receptor (pIgR). DNA sequence analysis of these clones provided the complete coding sequence of rat pIgR. Subsequent alignment of rat, rabbit and human predicted amino acid sequences demonstrated that the greatest degree of homology between the three pIgRs lies in their cytoplasmic tails; a region previously shown to be important for correct targetting and trancytosis of rabbit pIgR [(1984) Nature 308, 37-43].

Detergent enhances binding of a secreted HLA-A2 molecule to solid phase peptides.

We have constructed a secreted analogue (sA2) of the human class I molecule HLA-A2. sA2 was affinity purified both in the presence and absence of detergent and the effects of detergent on the magnitude and specificity of A2 binding to solid phase peptides tested. sA2 purified in the presence of detergent and detergent-solubilized A2 are shown to function comparably in the binding of the synthetic peptide M.Y + 57-68, a known T-cell epitope derived from the influenza A matrix protein. The molecules binding to M.Y + 57-68 typically represent 8% to 10% of the added protein. In contrast, less than 1% of sA2 protein purified in the absence of detergent binds M.Y + 57-68. This reduced binding is not due to a change in the affinity of sA2 for M.Y + 57-68. Addition of detergent at various stages of the purification and iodination procedures indicates that the longer the sA2 molecules are exposed to detergent the better they bind. However, the concentration of detergent during the actual binding assay does not appear to be critical. We also find that while the sA2-detergent and the sA2-no detergent molecules differ in the extent to which they bind various peptides, they do not differ in their patterns of binding. We conclude that detergent probably does not influence the specificity of class I/peptide binding but does increase the number of sA2 molecules that can participate in the binding of peptide either by generating and stabilizing "empty" sA2 molecules or by stabilizing a structure that is more amenable to binding peptide.

Aging effects on hepatic NADPH cytochrome P450 reductase, CYP2B1&2, and polymeric immunoglobulin receptor mRNAs in male Fischer 344 rats.

Aging perturbs the expression of many liver proteins, but the mechanisms remain unresolved. Expression of hepatic NADPH cytochrome P450 reductase, phenobarbital-induced CYP2B1&2, and the polymeric immunoglobulin receptor (pIgR) decline as a function of aging. We examined the effect of aging on the expression of the mRNA transcripts of these proteins, as well as those of alpha 2u-globulin and beta-actin in male F344 rats. Despite age-related losses in the expression of P450 reductase and plasma membrane-bound pIgR in the rat liver (approximately 30-50%), aging is is accompanied by 1) no change and 2) a modest decline (< 20%) in their respective mRNA steady state levels. On the other hand, the expression of phenobarbital-induced microsomal CYP2B1&2 and the steady state level of its mRNA exhibit parallel age-dependent shifts. The mRNA transcript for alpha 2u-globulin declines between maturity and old age, whereas the beta-actin mRNA level remains unchanged. These preliminary data are consistent with previous studies which suggest that aging may perturb hepatic CYP2B1&2 and alpha 2u-globulin at the transcriptional level, whereas changes in the expression of P450 reductase and pIgR may reflect posttranscriptional modifications.

Polymeric-Ig receptor gene expression in rabbit mammary gland during pregnancy and lactation: evolution and hormonal regulation.

The polymeric immunoglobulin receptor (poly Ig-R) mediates transcytosis of IgA and IgM antibodies produced by local plasma cells across epithelial cells of mucosal and glandular tissues. Gene expression of the poly-Ig R was analyzed in rabbit mammary gland during pregnancy and lactation. The poly Ig-R was expressed as early as day 8 (G8) of gestation and mRNA accumulation remained low until about G18. From G21, the mRNA abundance increased and reached steady state levels approximately 5-fold higher at day 15 of lactation (L15) when compared to basal levels at G8. The hormonal regulation of poly-Ig receptor gene expression was assessed in mammary organ cultures. Poly-Ig R mRNA accumulation in mammary explants cultured for 24 or 48 h in the presence of ovine prolactin (oPRL) was significantly increased to a maximal 4-fold level at 1 microgram ml-1 of oPRL. Estradiol (100 pg ml-1) or progesterone (1 microgram ml-1) did not further stimulate poly-Ig R expression. In contrast, their combination resulted in a significant 30-50% decrease of poly-Ig-R mRNA levels. The addition of 1 microgram ml-1 of cortisol to medium in the absence or presence of estradiol or progesterone decreased the amount of poly-Ig-R mRNA. The results suggest that until mid-pregnancy, poly-Ig-R expression is inhibited by elevated progesterone-estradiol concentrations and that the subsequent increase is due to the concomitant decrease of the two circulating steroids and the increase of serum prolactin levels.

The first exon of the human sc gene contains an androgen responsive unit and an interferon regulatory factor element.

Secretory component (SC) plays a key role in the transport of IgA and IgM to the lumina of many glands. The gene is constitutively expressed, but can be modulated by hormonal and immunological stimuli. Recently, the promoter and the first exon of the human sc gene have been cloned. The first exon contains a putative androgen/glucocorticoid response element (ARE/GRE) and an Interferon Regulatory Factor Element (IRF-E). Here we show that the ARE/GRE can bind the DNA-binding domain (DBD) of both the androgen (AR) and glucocorticoid receptor (GR) with a preference for the AR-DBD. In transient transfection experiments, this element confers higher responsiveness to androgens than to glucocorticoids. The IRF-E can function as an IRF-2, but surprisingly not as an IRF-I responsive element. We postulate that these two regulatory elements play a key role in the complex regulation of the sc gene in vivo.

Androgen control of secretory component mRNA levels in the rat lacrimal gland.

The purpose of this investigation was to determine whether the known gender-related differences in, and the endocrine control of, the production of secretory component (SC) by the rat lacrimal gland are associated with alterations in SC mRNA content. Levels of SC mRNA were measured in lacrimal tissues of intact, sham-operated, castrated, hypophysectomized, and testosterone-treated male and female adult rats by Northern blot procedures, which utilized a specific, [alpha-32P]-labelled rat SC cDNA probe. For control purposes, SC mRNA amounts were standardized to the beta-actin content in experimental blots. The location of SC mRNA in lacrimal glands was evaluated by in situ hybridization techniques, which involved exposure of tissue sections to sense or anti-sense [35S]-labelled SC RNA probes. Our results demonstrate that: (1) lacrimal glands of male rats contain a significantly greater amount of SC mRNA than those of female rats, and that this difference co-exists with distinct, gender-associated variations in the distribution of SC mRNA in lacrimal tissue; (2) orchiectomy or hypophysectomy, but not ovariectomy or sham surgery, leads to a marked decline in the lacrimal SC mRNA content; and (3) testosterone, but not placebo, administration to castrated male and female rats induces a significant increase in the SC mRNA levels in lacrimal tissue. Overall, these findings show that gender, androgens and the hypothalamic-pituitary axis exert a considerable influence on the SC mRNA content in the rat lacrimal gland.

Estradiol regulation of secretory component: expression by rat uterine epithelial cells.

Sex hormones are known to play an important role in the regulation of mucosal immunity in the female reproductive tract. The purpose of this study was to examine the effect of estradiol (E2) on secretory component (SC) expression by epithelial cells in the rat uterus and to determine whether SC mRNA is present in uterine tissues and is under hormonal control. When ovariectomized rats treated with E2 for 3 days and sacrificed 12 h after the last injection, expression of SC on luminal and glandular epithelial cells, as determined by immunohistochemistry, was elevated when compared to control animals. To determine whether E2 regulation of SC involves mRNA synthesis, uterine RNA was extracted and analyzed by Northern blot. These experiments demonstrated that SC RNA is present in uteri from intact rats and markedly increased when ovariectomized animals are treated with E2. In other studies, uterine epithelial cells from adult rats were isolated and grown on permeable membranes for 5 to 10 days. Under these conditions, isolated epithelial cells grow to confluence, form tight junctions, and preferentially secrete SC into the apical medium. These studies identify epithelial cells as a key target cell in the uterus for the regulation of mucosal immunity by E2, which we postulate will play an important role in studies to prevent and/or control the spread of sexually transmitted diseases.