Increased galectin-3 expression and intra-epithelial neutrophils in small airways in severe COPD.

Galectins-1 and -3 regulate epithelial proliferation/apoptosis and neutrophil activation, and are implicated in lung cancer and asthma. The role of galectins in chronic obstructive pulmonary disease (COPD), characterised by epithelial changes and neutrophil infiltration, remains unknown. In the present study, galectin-1 and -3 expression was assessed by immunohistology in the bronchial epithelium of lung specimens from eight severe COPD patients and compared with nine nonsmokers and six smokers without COPD. Findings were related to epithelial proliferation (Ki-67), tissue inflammation and lung function. Epithelial galectin-3 immunostaining was increased only in the small airways of COPD patients when compared with nonsmokers and smokers. In contrast, galectin-1 was only significantly increased in the small airways of the group of smokers. Ki-67+ epithelial cells and neutrophils were increased in the small airways of COPD patients when compared with smokers. Furthermore, intra-epithelial neutrophils correlated in the small airways with Ki-67+ epithelial cells and with the forced expiratory volume in one second/forced vital capacity ratio. However, no correlation was observed with galectin expression. The present study supports the hypothesis that distal airways represent an important site for detecting changes in chronic obstructive pulmonary disease. In patients with severe disease, an increased galectin-3 expression and neutrophil accumulation in the small airway epithelium was demonstrated, correlating with epithelial proliferation and airway obstruction.

Oxidative burst in lipopolysaccharide-activated human alveolar macrophages is inhibited by interleukin-9.

Interleukin (IL)-9 is known to regulate many cell types involved in T-helper type 2 responses classically associated with asthma, including B- and T-lymphocytes, mast cells, eosinophils and epithelial cells. In contrast, target cells mediating the effects of IL-9 in the lower respiratory tract remain to be identified. Therefore, the authors evaluated the activity of IL-9 on human alveolar macrophages (AM) from healthy volunteers. AM preincubated with IL-9 before lipopolysaccharide (LPS) stimulation exhibited a decreased oxidative burst, as previously shown with IL-4. The inhibitory effect of IL-9 was abolished by anti-hIL-9R alpha monoclonal antibody, and presence of IL-9 receptors on AM was demonstrated by immunofluorescence. Both IL-4 and IL-9 failed to modulate tumour necrosis factor-alpha, IL-8 and IL-10 release by LPS-stimulated AM. However, several observations suggested that IL-9 and IL-4 act through different mechanisms: 1) interferon-gamma antagonised the IL4- but not the IL-9-mediated inhibition of AM oxidative burst; 2) expression of CD14 was downregulated by IL-4 but not by IL-9 and 3) production of tumour growth factor-beta by activated AM was potentiated by IL-9 and not by IL4, and was required for the IL-9-mediated inhibition of AM oxidative burst. These observations provide additional information concerning the activity of interleukin-9 in the lung, related to inflammatory or fibrosing lung processes.

Lung mucosal immunity: immunoglobulin-A revisited.

Mucosal defence mechanisms are critical in preventing colonization of the respiratory tract by pathogens and penetration of antigens through the epithelial barrier. Recent research has now illustrated the active contribution of the respiratory epithelium to the exclusion of microbes and particles, but also to the control of the inflammatory and immune responses in the airways and in the alveoli. Epithelial cells also mediate the active transport of polymeric immunoglobulin-A from the lamina propria to the airway lumen through the polymeric immunoglobulin receptor. The role of IgA in the defence of mucosal surfaces has now expanded from a limited role of scavenger of exogenous material to a broader protective function with potential applications in immunotherapy. In addition, the recent identification of receptors for IgA on the surface of blood leukocytes and alveolar macrophages provides an additional mechanism of interaction between the cellular and humoral immune systems at the level of the respiratory tract.

Reduced epithelial expression of secretory component in small airways correlates with airflow obstruction in chronic obstructive pulmonary disease.

The epithelial polymeric immunoglobulin receptor/transmembrane secretory component (pIgR/SC) transports into secretions polymeric immunoglobulin A (pIgA), which is considered the first line of defense of the respiratory tract. The present study, done with quantitative immunohistochemistry, evaluated epithelial expression of secretory component (SC) and Clara cell protein (CC16) and neutrophil infiltration into the airways of eight patients with severe chronic obstructive pulmonary disease (COPD) who were undergoing lung transplantation, as compared with these processes in six nonsmoking patients with pulmonary hypertension who were used as controls and in lung specimens from five smokers without chronic bronchitis. Staining for SC was significantly decreased in the COPD patients as compared with the controls, both in large (mean optical density [MOD]: 23.4 [range: 21.1 to 27.8] versus 42.2 [range: 28.2 to 49.3], p = 0.003) and in small airways (MOD: 30.8 [range: 20.3 to 39.4] versus 41.5 [range: 39.2 to 46.2], p = 0.003). SC expression in small airways correlated strongly with functional parameters such as FEV1 (Kendall's tau (K) = 0.76, p = 0.008), FVC (K = 0.64, p = 0.03), and midexpiratory flow at 50% of VC (MEF50) (K = 0.74, p = 0.01). The reduced expression of SC in large airways correlated with neutrophil infiltration in submucosal glands (K = -0.47, p = 0.03). Expression of CC16 in the bronchial epithelium of COPD patients was also significantly decreased as compared with that of controls, especially in small airways (MOD: 28.3 [range: 26.8 to 32.4] versus 45.8 [range: 40.7 to 56.0], p = 0.002), but no correlation was observed with lung function tests. In conclusion, this study shows that reduced expression of SC in airway epithelium is associated with airflow obstruction and neutrophil infiltration in severe COPD.

Polymeric IgA binding to the human pIgR elicits intracellular signalling, but fails to stimulate pIgR-transcytosis.

The intracellular pathway of polymeric immunoglobulin receptor (pIgR) is governed by multiple signals that lead to constitutive transcytosis. In addition, in transfected polarized MDCK cells, polymeric immunoglobulin A (pIgA) binding stimulates rabbit pIgR-transcytosis, owing to phospholipase-C gamma 1 activation and increase of intracellular calcium. Transcytosis of rat pIgR across hepatocytes is similarly accelerated by pIgA injection. In contrast we show here that human Madrin-Darby Canine Kidney (pIgR)-transcytosis, in human Calu-3 and human pIgR-transfected MDCK cells, is not promoted by pIgA, as monitored by a continuous apical release of its secreted ectodomain. However, the incubation of cells expressing human or rabbit pIgR with pIgA induces a comparable IP3 production, and pIgR-transcytosis of either species is accelerated by the protein kinase C (PKC)-activator phorbol myristate acetate. Without pIgA, mimicking phospholipase-C activation by combining low concentrations of phorbol myristate acetate with ionomycin, or high concentrations of ionomycin alone, stimulates the rabbit, but not the human, pIgR transcytosis. These data suggest that the species difference in pIgA-induced pIgR-transcytosis does not stem from the defective production of second messengers, but from a different sensitivity of pIgR to intracellular calcium. Our results outline the danger of extrapolating to humans the abundant data obtained from mucosal vaccination of laboratory animals.

Secretory mucosal immune mechanisms.

Mucosal immune mechanisms in the airways are the first specific line of defense, protecting the body from pathogens. The respiratory epithelium actively transports locally produced dimeric IgA in the respiratory secretions by transcytosis, through the pIgR. S-IgA production therefore requires epithelial integrity. S-IgA at the epithelial level is active in several non-inflammatory pathways including intracellular neutralization of virus, antigen excretion, binding to bacterial adhesins. Local IgA production is regulated by various growth factors and cytokines of both epithelial and non-epithelial origin. The respiratory epithelium is thought to play a crucial role in this process. In addition, in chronic airway inflammation, IgA production demonstrates a correlation with eosinophil activation both in vitro and in vivo. While increased IgA and S-IgA production is reported in asthmatics, decreased SC production has been documented in CF and COPD patients, further impairing their local defense mechanisms.

Species-specific binding of human secretory component to SpsA protein of Streptococcus pneumoniae via a hexapeptide motif.

SpsA, a pneumococcal surface protein belonging to the family of choline-binding proteins, interacts specifically with secretory immunglobulin A (SIgA) via the secretory component (SC). SIgA and free SC from mouse, rat, rabbit and guinea-pig failed to interact with SpsA indicating species-specific binding to human SIgA and SC. SpsA is the only pneumococcal receptor molecule for SIgA and SC as confirmed by complete loss of SIgA and SC binding to a spsA mutant. Analysis of recombinant SpsA fusion proteins showed that the binding domain is located in the N-terminal region of SpsA. By the use of different truncated N-terminal SpsA fusion proteins, the minimum binding domain was shown to be composed of 112 amino acids (residues 172-283). The sequence of this 112-amino-acids domain was used to spot synthesize 34 overlapping peptides, consisting of 15 amino acids each, with an offset of three amino acids on a cellulose membrane. One of the peptides reacted specifically with both SIgA and SC. By using a second membrane with immobilized synthetic peptides of decreasing length containing parts of the identified 15-amino-acid motif a hexapeptide, YRNYPT was identified as the binding motif for SC and SIgA. SpsA proteins with a size smaller than the assay-positive domain of 112 amino acids were able to inhibit the interaction of SIgA and pneumococci provided they contained the binding motif. The results indicated that the hexapeptide YRNYPT located in SpsA of pneumococcal strain type 1 (ATCC 33400) between amino acids 198 and 203 is involved in SIgA and SC binding. Because synthetic peptides containing only parts of the hexapeptide also assayed positive, these results further suggest that at least the amino acids YPT of the identified hexapeptide are critical for binding to SC and SIgA. Amino acid substitutions in the identified putative binding motif abolished SC-/SIgA-binding activity of the mutated SpsA protein, confirming the functional activity of this hexapeptide and the critical role of the amino acids YPT in SC and SIgA binding. Identification of this motif, which is highly conserved in SpsA protein among different serotypes, might contribute towards a new peptide based vaccine strategy.

The SRC family protein tyrosine kinase p62yes controls polymeric IgA transcytosis in vivo.

Transcytosis of polymeric immunoglobulin A (pIgA) across epithelial cells is mediated by the polymeric immunoglobulin receptor (pIgR). Binding of pIgA to pIgR stimulates transcytosis of the pIgA-pIgR complex via a signal transduction pathway that is dependent on a protein tyrosine kinase (PTK) of the SRC family. Here we identify the PTK as p62yes. We demonstrate the specific physical and functional association of the pIgR with p62yes in rodent liver. Analysis of p62yes knockout mice revealed a dramatic reduction in the association of tyrosine kinase activity with the pIgR and in transcytosis of pIgA. We conclude that p62yes controls pIgA transcytosis in vivo.

Lack of SC/pIgR-mediated epithelial transport of a human polymeric IgA devoid of J chain: in vitro and in vivo studies.

Three human polymeric IgA (pIgA) myeloma proteins of tetrameric size were compared for their J-chain content, their in vitro secretory component (SC)-binding ability, and their capacity to be transcytosed by polymeric immunoglobulin receptor (pIgR)-expressing epithelial cells in vitro and rat hepatocytes in vivo. One of the three pIgA preparations, pIgA-L, was shown to lack J chain and was unable to combine with purified free human and rat SC, whereas pIgA-G and pIgA-C contained J chain and combined readily with SC. Furthermore, pIgA-L was not transferred into rat bile after intravenous injection, and was hardly transported apically by polarized Madin-Darbey canine kidney cell monolayers expressing the human pIgR, whereas pIgA-G and pIgA-C were efficiently transported in both test systems. Together with our recent demonstration that antibodies to human J chain block the SC/pIgR-mediated epithelial transport of pIgA, these data unanimously confirm the proposed key role of J chain in the epithelial transport of polymeric immunoglobulins into exocrine secretions.

Molecular maturation and functional expression of mouse polymeric immunoglobulin receptor.

Mouse polymeric immunoglobulin receptor (pIgR) cDNA was stably introduced into a hamster-derived fibroblastic cell line, Chinese hamster ovary (CHO) cell, by the calcium phosphate method. Surface expression of pIgR was detected by immunostaining and FACS analysis. The immunoprecipitated products of cell lysates revealed that the molecular mass of the most mature form of pIgR was approximately 120 kDa. Western blotting and metabolic labeling experiments followed by immunoprecipitation with an anti-mouse secretory component (SC) Ab demonstrated the existence of a 110 kDa immature form of pIgR. The reason for the existence of two forms of pIgR molecule was examined by conducting pulse-chase experiments which revealed the pIgR underwent molecular maturation. During this process, the 110 kDa form of pIgR was converted into a 120 kDa form by glycosylation. Moreover, tunicamycin treatment revealed the core form of pIgR had a molecular mass of approximately 100 kDa. The pIgR expressed on the surface of the transfectant could specifically bind and take up mouse polymeric IgA (MOPC 315), suggesting that, at least in this mouse system, cell type-specific molecules are not necessary for surface pIgR expression and polymeric immunoglobulin (pIg) binding and uptake.

Secretory component production by human bronchial epithelial cells is upregulated by interferon gamma.

Secretory immunoglobulin A (S-IgA) participates in the first noninflammatory line of defence of the respiratory tract. S-IgA consists of dimeric IgA (dIgA) produced by plasma cells and secretory component (SC) produced by epithelial cells. This study compared SC production by primary cultures of human bronchial epithelial cells (HBEC) and by respiratory epithelial cell lines. Among the cell lines, A549 did not produce detectable SC, 16HBE produced very low levels of SC, while CALU-3 produced significant levels of SC. HBEC produced SC in nonpolarized and polarized primary cultures, where it was secreted apically. Polarized HBEC transcytosed radiolabelled and cold dIgA, resulting in the presence of S-IgA in their apical media. SC production and IgA transcytosis by polarized HBEC were upregulated by interferon-gamma (IFN-gamma) after 48 h. By reverse transcription-polymerase chain reaction, no SC messenger ribonucleic acid (mRNA) was detected in A549 and 16HBE, while SC mRNA in CALU-3 was comparable to that of HBEC incubated for 48 h with IFN-gamma. By immunocytochemistry, HBEC expressed SC immunostaining and its intensity increased after 48 h with IFN-gamma. It is concluded that human bronchial epithelial cells produce secretory component and transcytose dimeric immunoglobulin A in vitro. These processes were apically polarized and upregulated by interferon-gamma. Among the cell lines studied, only CALU-3 expressed secretory component-messenger ribonucleic acid and produced detectable secretory component.

In vivo stimulation of polymeric Ig receptor transcytosis by circulating polymeric IgA in rat liver.

Binding of human polymeric IgA ligand to its epithelial cell polymeric Ig receptor, pIgR, has been shown to stimulate pIgR apical transcytosis in an in vitro system, based on polarized confluent MDCK cells expressing rabbit pIgR. The present study aimed at testing whether such a stimulation also occurs in vivo. Transcytosis of pIgR was monitored by rat liver output of total secretory component (SC) into bile, measured by radial immunodiffusion as the sum of free SC and pIgA-bound SC. Whereas in the perfused rat liver system addition of pIgA to the perfusate showed no effect, i.v. injection of human and rat pIgA, but not of monomeric IgA nor PBS, in living rats significantly increased total bile SC output for more than 1 h. Furthermore, depletion of the normal pIgA level circulating in the liver before injecting more pIgA was not required to show the stimulation. Our data thus strongly suggest that stimulation of liver pIgR transcytosis by pIgA ligand binding is physiologically relevant, helping to quickly adjust pIgA transport into bile to increase circulating pIgA levels, without need for increased SC/pIgR synthesis.

Primary rat lacrimal cells undergo acinar-like morphogenesis on reconstituted basement membrane and express secretory component under androgen stimulation.

Single cells or small cell clusters, isolated from the rat lacrimal gland, were incubated on reconstituted basement membrane (matrigel) in a well-defined serum-free medium. During the first days of culture, cells reassociated and reorganized in structures resembling acini. These multicellular structures, maintained in culture for 2 weeks, consisted of well-polarized cuboidal cells surrounding a central lumen and exhibiting apically located microvilli. Myoepithelial cells were observed at the periphery of the acinar structures. Both in the native lacrimal and in the cultured aggregates, epithelial cells displayed strong immunoreactivity for cytokeratin 8, while myoepithelial cells were immunoreactive for vimentin and alpha-smooth muscle isoactin. These data indicate that the cultured aggregates closely mimic the in vivo architecture of lacrimal glands both by morphology and immunohistochemistry. We further demonstrated the presence of an intact androgen receptor and the ability of the cultured aggregates to respond to androgens with increased secretion of the secretory component. Comparable androgen responses were observed in lacrimal gland cultures of 5-week-old male and female rats. In conclusion, we report a morphologically and functionally differentiated culture system of primary rat lacrimal cells, in which androgen-regulated gene expression was observed. This culture model provides a unique experimental paradigm for studying the effects of hormones, cytokines, and growth factors on the morphogenesis, growth, and functional differentiation of lacrimal glands.

Antibody against the human J chain inhibits polymeric Ig receptor-mediated biliary and epithelial transport of human polymeric IgA.

To emphasize the requirement for a J chain in native polymeric immunoglobulins for their selective transport into exocrine secretions, IgG, purified from two different antisera specific for the human J chain, was shown to: (i) bind in vitro to human polymeric IgA (pIgA) by density gradient ultracentrifugation; (ii) inhibit binding in vitro of rat secretory component to human pIgA; (iii) inhibit hepatic transport of human pIgA into rat bile in vivo; and (iv) inhibit apical transcytosis of pIgA in vitro by polarized human polymeric immunoglobulin receptor (pIgR)-expressing Madin-Darby canine kidney cells. Inhibition of biliary transport increased with the molar ratio of anti-J chain antibodies against pIgA and their incubation time. Anti-J chain F(ab')2 and Fab fragments also inhibited biliary transport, excluding a role for phagocytic clearance or excessive size of the immune complexes. Anti-human-Fc alpha Fab, bound to human pIgA in complexes of larger size than those with anti-J chain Fab, did not inhibit biliary transport of human pIgA. Propionic acid-denatured human pIgA, although containing J chains, was very poorly transported into rat bile. Altogether, our data strongly support, now also by in vivo experiments, the crucial role of the J chain of native pIgA in its selective pIgR-mediated transport into secretions, as suggested long ago by in vitro data only. Recent data on J chain-knockout mice, with low IgA levels in bile and feces, cannot explain the role of the J chain in contributing to the secretory component/pIgR-binding site of normal pIgA, but otherwise agree with our study.

Differential effects of the T helper cell type 2-derived cytokines IL-4 and IL-5 on ligand binding to IgG and IgA receptors expressed by human eosinophils.

Increased numbers of eosinophilic granulocytes that exhibit an activated phenotype are found in bronchial tissue and bronchial alveolar lavage fluid of patients with allergic asthma. Little is known about the processes that lead to activation of eosinophils in vivo, but Igs might be important stimulants. In the present study we investigated the capacity of human eosinophils to interact with beads coated with human serum IgG or IgA. Binding of IgG/IgA-coated beads to eosinophils from normal donors appeared to be dependent on priming with Th2-derived cytokines. Priming with granulocyte-macrophage CSF, IL-4, or IL-5 is required for eosinophils to form rosettes with IgA-beads. IL-4 priming resulted in a fast and transient effect on binding of IgA-beads, whereas the effect of IL-5 priming was slower and longer lasting. The expression of Fc alphaR (CD89) was low compared with that on neutrophils, and experiments with the blocking mAb My43 (CD89) showed no inhibition of rosette formation between eosinophils and IgA-coated beads. However, polymeric myeloma IgA effectively inhibited the rosette formation of IgA-coated beads to eosinophils. Binding of IgG-beads could only be primed with granulocyte-macrophage CSF and IL-5, not with IL-4. These data are concurrent with the hypothesis that Th2-derived cytokines spatially produced at the side of an allergic inflammatory response can direct eosinophils to a rather restricted primed phenotype by IL-4 or to a more generalized primed phenotype by IL-5.

Effects of intrajejunal perfusion and chronic ingestion of Lactobacillus johnsonii strain La1 on serum concentrations and jejunal secretions of immunoglobulins and serum proteins in healthy humans.

OBJECTIVES: Link-Amster reported an increase in serum IgA when healthy subjects ingested a fermented dairy product containing Lactobacillus johnsonii La1. We aimed to assess the effects of La1 on the jejunal secretions and serum concentrations of total and specific immunoglobulins and proteins. METHODS: Twelve healthy volunteers ingested a fermented milk containing La1 or a control from day 1 till day 28, following a randomised double blind protocol. At days 0 and 28, the jejunum was successively perfused with a control solution and with a La1 suspension. The serum concentrations and jejunal secretions of albumin, orosomucoid, transferrin, alpha 2-macroglobulin, m-IgA, p-IgA, IgG, IgM, secretory component, and specific antibodies against La1 were assessed. RESULTS: Serum concentrations of IgA slightly increased between d0 and d28 in the group receiving La1 (1.85 +/- 0.64 g/L vs 1.76 +/- 0.76; P = 0.02). The other parameters were not altered. CONCLUSION: This study shows that the immunomodulating effects of La1 ingestion in man are not due to modification of jejunal protein permeability.

Human colonic intraepithelial lymphocytes regulate the cytokines produced by lamina propria mononuclear cells.

Using an in vitro autologous human system, the immunomodulatory function of colonic intraepithelial lymphocytes (IEL) on cytokine production by lamina propria mononuclear cells (LPMNC) has been investigated. In contrast to LPMNC, colonic IEL produced only low amounts of IL-10, interferon-gamma and interleukin-2. However, co-culture experiments (IEL + LPMNC) have shown that IEL can enhance the PHA-induced synthesis of IL-2 and interferon-gamma, but not IL-10 by LPMNC. Using a transwell filter culture system apparatus, this effect was shown not to require a cell-to-cell interaction. Thus, IEL in vitro may modulate the cytokine synthesis of LPMNC, through the production of soluble factors. This may prove highly relevant in the in vivo immune activation of the gastrointestinal mucosa.

Hepatobiliary transport of IgA in the golden Syrian hamster (Mesocricetus auratus).

Do hamsters, like rats, rabbits and mice, possess an hepatocyte 'IgA pump' whereby circulating plasma polymeric IgA (pIgA) is actively transported into bile, against a concentration gradient, via the polymeric Ig receptor or secretory component (SC)? Precipitating antisera, raised against rat Igs and serum proteins, and crossreacting with their hamster homologues, detected hamster SC by immunoelectrophoresis in bile, but not serum. Gel filtration of hamster bile indicated that free SC eluted between IgG and albumin, as for other mammals. Hamster bile IgA was pIgA, and was true secretory IgA (SIgA) by its reaction with anti-SC antiserum and by SDS-PAGE with reduction. Hamster serum IgA comprised both pIgA and IgA monomers. Mean bile-to-serum concentration ratios (B/S) for IgA, IgG, transferrin and albumin, measured by radial immunodiffusion, were 2.65, 0.019, 0.024, and 0.016, respectively, demonstrating strongly selective enrichment of bile in IgA. Human 125I-labelled dimeric IgA was injected into the circulation of five hamsters with cannulated bile ducts; 20% of the [125I]IgA (> 95% precipitable by trichloroacetic acid) was recovered in bile within 5 h, a figure close to that for mice, but smaller than that for rats and rabbits. The data suggest that bile significantly contributes to hamster intestinal SIgA, as shown for rats, rabbits and mice. This could be relevant to studies where hamsters are used as an experimental model for infection by the human intestinal pathogen, Clostridium difficile.

Contribution of serum IgA to intestinal lymph IgA, and vice versa, in minipigs.

Immune cells in pig gut lymph are rather well studied, but data on gut lymph immunoglobulins and their origin are nonexistent. Such data are important to understand the interplay between pig systemic and intestinal immunity as a basis for vaccination studies. In some species, gut lymph contributes much to plasma IgA, but apparently not in humans. To estimate the contributions of pig serum IgA to intestinal lymph IgA and vice versa, concentrations of IgA, IgG, IgM, albumin, haptoglobin, C3 and alpha 2-macroglobulin were measured by radial immunodiffusion in paired porcine intestinal lymph and serum samples. All proteins, except IgA, had lymph/serum ratios (< 1.0) inversely related to their size, depending on passive diffusion from serum. The mean lymph/serum ratio of IgA was 2.2 instead of an expected 0.50 or 0.65 (dimer or monomer, respectively), indicating that of the IgA in gut lymph, 22.7 or 29.5% came from serum, vs 77.3 or 70.5% from the intestine. Percentage of polymeric IgA, measured by gelfiltration and corrected radial immunodiffusion, was 64.3% in porcine mesenteric lymph and 47.3% in serum. As the pig plasma volume and daily gut lymph flow into circulation were known, it could be calculated that roughly 31% of the total plasma IgA originated daily from local intestinal synthesis, reaching blood via mesenteric lymph.