Interleukin-22 protects against non-typeable Haemophilus influenzae infection: alteration during chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease is a major health problem becoming a leading cause of morbidity and mortality worldwide. A large part of these disorders is associated with acute exacerbations resulting from infection by bacteria, such as non-typeable Haemophilus influenzae (NTHi). Our understanding of the pathogenesis of these exacerbations is still elusive. We demonstrate herein that NTHi infection of mice chronically exposed to cigarette smoke (CS), an experimental model of chronic obstructive pulmonary disease (COPD), not only causes acute pulmonary inflammation but also impairs the production of interleukin (IL)-22, a cytokine with potential anti-bacterial activities. We also report that mice lacking IL-22, as well as mice exposed to CS, have a delayed clearance of NTHi bacteria and display enhanced alveolar wall thickening and airway remodeling compared with controls. Supplementation with IL-22 not only boosted bacterial clearance and the production of anti-microbial peptides but also limited lung damages induced by infection both in IL-22-/- and CS-exposed mice. In vitro exposure to CS extract altered the NTHi-induced IL-22 production by spleen cells. This study shows for the first time that a defect in IL-22 is involved in the acute exacerbation induced by NTHi infection during experimental COPD and opens the way to innovative therapeutic strategies.

Binding sites of leukocyte beta 2 integrins (LFA-1, Mac-1) on the human ICAM-4/LW blood group protein.

The red cell ICAM-4/LW blood group glycoprotein, which belongs to the family of intercellular adhesion molecules (ICAMs), has been reported to interact with CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) beta(2) integrins. To better define the basis of the ICAM-4/beta(2) integrin interaction, we have generated wild-type, domain-deleted and mutated recombinant chimeric ICAM-4-Fc proteins and analyzed their interaction in a cellular adhesion assay with LFA-1 and Mac-1 L-cell stable transfectants. We found that monoclonal antibodies against CD11a, CD11b, CD18, or LW(ab) block adhesion of transfectant L-cells to immobilized ICAM-4-Fc protein and that the ICAM-4/beta(2) integrin interaction was highly sensitive to the presence of the divalent cations Ca(2+) and Mg(2+). Deletion of individual Ig-domains D1 or D2 of the extracellular part of ICAM-4 showed that LFA-1 binds to the first Ig-like domain, whereas the Mac-1 binding site encompassed both the first and the second Ig-like domains. Based on the crystal structure of ICAM-2, we propose a model for the Ig-like domains D1 and D2 of ICAM-4. Accordingly, by site-directed mutagenesis of 22 amino acid positions spread out on all faces of the ICAM-4 molecule, we identified four exposed residues, Leu(80), Trp(93), and Arg(97) on the CFG face and Trp(77) on the E-F loop of domain D1 that may contact LFA-1 as part of the binding site. However, the single and double mutants R52E and T91Q on the CFG face of domain D1, which correspond to the key residues Glu(34) and Gln(73) for ICAM-1 binding to LFA-1, had no effect on LFA-1 binding. In contrast, all mutants on the CFG face of domain D1 and residues Glu(151) and Thr(154) in the C'-E loop of the domain D2 seem to play a dominant role in Mac-1 binding. These data suggest that the binding site for LFA-1 on ICAM-4 overlaps but is distinct from the Mac-1 binding site.

Characterization of the gene encoding the human LW blood group protein in LW+ and LW- phenotypes.

The LW blood group is carried by a 42-kD glycoprotein that belongs to the family of intercellular adhesion molecules. The LW gene is organized into three exons spanning an HindIII fragment of approximately 2.65 kb. The exon/intron architecture correlates to the structural domains of the protein and resembles that of other Ig superfamily members except that the signal peptide and the first Ig-like domain are encoded by the first exon. The 5'UT region (nucleotides -289 to +9) includes potential binding sites for various transcription factors (Ets, CACC, SP1, GATA-1, AP2) and exhibited a significant transcriptional activity after transfection in the erythroleukemic K562 cells. No obvious abnormality of the LW gene, including the 5'UT region, has been detected by sequencing polymerase chain reaction-amplified genomic DNA from RhD+ or RhD- donors and from an Rhnull variant that lacks the Rh and LW proteins on red blood cells. However, a deletion of 10 bp in exon 1 of the LW gene was identified in the genome of an LW (a- b-) individual (Big) deficient for LW antigens but carrying a normal Rh phenotype. The 10-bp deletion generates a premature stop codon and encodes a truncated protein without transmembrane and cytoplasmic domain. No detectable abnormality of the LW gene or transcript could be detected in another LW(a- b-) individual (Nic), suggesting the heterogeneity of these phenotypes.

The red cell LW blood group protein is an intercellular adhesion molecule which binds to CD11/CD18 leukocyte integrins.

Leukocyte adhesion involves the leukocyte-specific integrins CD11a/CD18, CD11b/CD18 and CD11c/CD18, which bind to intercellular adhesion molecules (ICAM). Three ICAM have been described, and are expressed on leukocytes and various other cells, but are absent from red cells. Here, we show that the red cell Landsteiner-Wiener (LW) blood group glycoprotein is an ICAM which binds to the leukocyte-specific integrins. This finding has important implications in red cell physiology.

Molecular basis and expression of the LWa/LWb blood group polymorphism.

The Landsteiner-Wiener (LW) blood group antigens reside on a 42-kD erythrocyte membrane glycoprotein that has recently been cloned. Here, we found that the molecular basis for the LWa/LWb polymorphism is determined by a single base pair mutation (A308G) that correlates with a Pvu II restriction site and results in a Gln70Arg amino acid substitution. COS-7 cells transfected with LWa or LWb cDNAs reacted with human anti-LWa and anti-LWb sera, respectively, as well as with a murine monoclonal anti-LWab antibody, as shown by flow cytometry analysis. Moreover, a 42-kD protein was immunoprecipitated from the transfected cells with the monoclonal anti-LWab antibody. These findings indicate that LWa and LWb are alleles of the LW blood group locus as defined also by a monoclonal anti-LWab of nonhuman origin. In addition, the LW locus has been assigned to chromosome 19p13.3 by in situ hybridization. Study by Southern blot analysis indicated also that the LW locus is composed of a single gene that was not grossly rearranged in rare LW(a-b-) and Rhnull individuals deficient for LW antigens. In addition, Pvu II restriction fragment-length polymorphism analysis indicated that these variants were all homozygous for a phenotypically silent LWa allele.

The LW blood group glycoprotein is homologous to intercellular adhesion molecules.

The LW blood group antigens reside on a 42-kDa erythrocyte membrane glycoprotein that was purified by immunoaffinity and partially sequenced. From this information, a specific PCR-amplified DNA fragment was used to screen a lambda gt11 human bone marrow cDNA library. Two forms of cDNA were isolated; the first encoded a single spanning transmembrane protein of 270 amino acids, including a 29-amino acid peptide signal and four potential N-glycosylation sites, and the second encoded a shortened protein form of 236 residues devoid of transmembrane and cytoplasm domains. A rabbit antibody raised against the 15 N-terminal amino acids of the predicted protein reacted on immunoblots with authentic LW glycoprotein and in indirect agglutination test with all human erythrocytes except those from LW(a-b-). This showed that the protein encoded by these clones was LW gene product and suggested that the N terminus of the LW protein is oriented extracellularly. Most interestingly, the LW protein was found to exhibit sequence similarities (with approximately 30% identity) with intercellular adhesion molecules ICAM-1, -2, and -3, which are the counter-receptors for the lymphocyte function-associated antigens LFA-1. The extracellular domain of LW consists, like that of ICAM-2, of two immunoglobulin-like domains, and the critical residues involved in the binding of LFA-1 to ICAMs were partially conserved in LW.

Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides.

Rabbit polyclonal antibodies were raised against synthetic peptides corresponding to hydrophilic regions of the human Rhesus (Rh) IX cDNA-encoded polypeptide predicted to be extracellularly or intracellularly exposed in the topologic model of the Rh blood group protein. Four antibodies encompassing residues 33-45 (MPC1), 224-233 (MPC4), 390-404 (MPC6), and 408-416 (MPC8) were characterized and compared with a polyclonal anti-Rh protein obtained by immunization with purified Rh proteins. All antibodies had specificity for authentic Rh polypeptides and reacted on Western blot with Rh proteins immunoprecipitated with human monoclonal anti-RhD, -c, and -E. MPC1, but not the other antibodies, agglutinated all human erythrocytes except Rhnull and Rhmod cells, which either lack totally or are severely deficient in Rh proteins, respectively. Immunoblotting analysis with membrane proteins from common and rare variants showed that MPC1 and MPC8 reacted in Western blot with 32-Kd Rh polypeptides from all common red blood cells except those from Rhnull and Rhmod, indicating that peptide regions 33-45 and 408-416 may be common to several if not all Rh proteins, whatever the Rh blood group specificity. MPC4 reacted only with membrane preparations from cells carrying the E antigen, whereas MPC6 recognized preferentially the Rh proteins from E and Ee preparations, suggesting that the protein encoded by the RhIXb cDNA carries the E and/or e antigen(s). Immunoadsorption experiments using inside-out or right-side-out sealed vesicules from DccEE red blood cells as competing antigen showed that the MPC6 and MPC8 antibodies bound only to the cytoplasmic side of the erythrocyte membrane, thus providing evidence for the intracellular orientation of the C-terminal 27 residues of the Rh polypeptides. Attempts to transiently or stably express the Rh polypeptides. Attempts to transiently or stably express the Rh cDNA in eukaryotic cells were largely unsuccessful, suggesting that Rh antigen expression at the cell surface requires correct transport and/or folding of the Rh proteins, possibly as a complex with one-membrane proteins of the Rh cluster that are lacking in Rhnull cells.

Surface orientation and antigen properties of Rh and LW polypeptides of the human erythrocyte membrane.

Time course digestion of intact human erythrocytes and right side-out vesicles with carboxypeptidase Y altered the Rh polypeptides and removed the 125I label that is normally incorporated by cell-surface radioiodination, but did not affect the RhD, Rhc, or RhE antigens. Under the same conditions, however, the LW antigens were rapidly destroyed. Digestion of inside-out and right side-out vesicles with aminopeptidase M was without any detectable effect on the Rh and LW antigens or polypeptides, although glycophorin A was degraded from right side-out but not from inside-out vesicles. These findings demonstrate that the C-terminal domain of the Rh and LW polypeptides is exposed at the external surface of human erythrocytes and indicate, in addition, that the LW antigens and tyrosine residue(s) of the LW and Rh proteins, respectively, are located close to the C termini of these polypeptides. Further studies using monoclonal and polyclonal antibodies showed that LW antigen expression is inhibited by treatment of red cells with EDTA and is selectively restored by Mg2+, but not by Mn2+ or Ca2+, whereas the Rh antigens were not affected under these conditions. In addition, O- and N-glycanase digestion of the LW glycoprotein removed its sugar chains, but did not alter significantly the epitopes recognized by the monoclonal anti-LW antibody.

Oral immunization of mice with a live recombinant Yersinia enterocolitica O:9 strain that produces the cholera toxin B subunit.

The 70-kilobase pYV plasmid of Yersinia enterocolitica encodes a set of proteins called Yops that are produced during infection. To use Y. enterocolitica as a live carrier to present the cholera toxin B (CT-B) subunit to the immune system, we constructed an operon fusion between ctxB and the yop51 gene. This operon fusion was either cloned on an RSF1010-derived plasmid or integrated into the pYV plasmid itself. In Y. enterocolitica, both constructions directed the synthesis of free CT-B only under conditions of Yops production, i.e., at 37 degrees C in a medium deprived of Ca2+. Bacteria containing both types of recombinant plasmids were given orally to mice. A serum antibody response against CT-B was detected in both cases. A secretory immunoglobulin A activity specific to CT-B was also observed in the intestinal secretions. According to immunoblot analysis, the serum antibody response was only directed against the polymeric form of the B subunit. The ctxB gene was also inserted in frame within yop51, giving a chimeric Yop51-CT-B protein that was secreted into the surrounding medium. In this case, however, no antibody response was observed after oral inoculation of mice. This lack of response probably results from the inability of the hybrid protein to assemble into the polymeric form of the B subunit.

Molecular cloning and protein structure of a human blood group Rh polypeptide.

cDNA clones encoding a human blood group Rh polypeptide were isolated from a human bone marrow cDNA library by using a polymerase chain reaction-amplified DNA fragment encoding the known common N-terminal region of the Rh proteins. The entire primary structure of the Rh polypeptide has been deduced from the nucleotide sequence of a 1384-base-pair-long cDNA clone. Translation of the open reading frame indicates that the Rh protein is composed of 417 amino acids, including the initiator methionine, which is removed in the mature protein, lacks a cleavable N-terminal sequence, and has no consensus site for potential N-glycosylation. The predicted molecular mass of the protein is 45,500, while that estimated for the Rh protein analyzed in NaDodSO4/polyacrylamide gels is in the range of 30,000-32,000. These findings suggest either that the hydrophobic Rh protein behaves abnormally on NaDodSO4 gels or that the Rh mRNA may encode a precursor protein, which is further matured by a proteolytic cleavage of the C-terminal region of the polypeptide. Hydropathy analysis and secondary structure predictions suggest the presence of 13 membrane-spanning domains, indicating that the Rh polypeptide is highly hydrophobic and deeply buried within the phospholipid bilayer. In RNA blot-hybridization (Northern) analysis, the Rh cDNA probe detects a major 1.7-kilobase and a minor 3.5-kilobase mRNA species in adult erythroblasts, fetal liver, and erythroid (K562, HEL) and megakaryocytic (MEG01) leukemic cell lines, but not in adult liver and kidney tissues or lymphoid (Jurkat) and promyelocytic (HL60) cell lines. These results suggest that the expression of the Rh gene(s) might be restricted to tissues or cell lines expressing erythroid characters.

Murine monoclonal antibodies against a unique determinant of erythrocytes, related to Rh and U antigens: expression on normal and malignant erythrocyte precursors and Rhnull red cells.

Three murine monoclonal antibodies (Mabs) MB-2D10, LA-18.18 and LA-23.40 were prepared. They reacted with red cells of all common and most rare blood-group phenotypes, with the exception of those of the RhnullU negative and RhmodU negative phenotypes. So far, only a single example of an alloantibody (Duclos or anti-Rh38) of a similar specificity has been found. Serological studies indicated that the Mabs were probably not directed against an antigenic determinant of Rh polypeptides, the LWab glycoprotein or glycophorin B, all structures absent from or aberrantly expressed on Rhnull red cells. The antigen was found to be erythrocyte-specific, and was also present on pro-erythroblasts, erythroblasts and malignant erythroblastoid cells but not on erythroid progenitors in the bone marrow. The Mabs were found to block each other in an immune rosette method and are thus probably directed against the same epitope or against neighbouring epitopes on the same structure. In immunochemical studies, MB-2D10 precipitated the 30-32 kDa Rh polypeptides from red cell membranes and a protein or proteins which formed diffuse and overlapping bands in SDS-polyacrylamide gel electrophoresis, with Mrs of 40-200 kDa (probably the Rh-related glycoproteins). Under certain experimental conditions glycophorin B appeared to be coprecipitated. The 2D10 structure, detected by the Mabs, seems to be part of a complex of proteins and/or glycoproteins, which includes Rh polypeptides, the LWab glycoprotein and glycoproteins recognized by various Mabs with Rh-related specificities. In the red cell membrane, the complex may be associated with glycophorin B.

Comparative analysis by two-dimensional iodopeptide mapping of the RhD protein and LW glycoprotein.

The RhD polypeptide and LW glycoprotein were separately immunopurified with monoclonal antibodies and compared by two-dimensional (2-D) iodopeptide mapping after digestion with alpha-chymotrypsin. These proteins have distinct 2-D maps, as seen after 125I-labeling tyrosine residues (chloramine-T procedure), and even more strikingly after labeling primary amine residues (Bolton-Hunter procedure). Of the more than 20 iodopeptides visualized, only five migrated identically when preparations of RhD and LW were directly compared, suggesting that RhD and LW are different proteins that may share some common protein domains. N-glycanase treatment of the iodopeptides did not modify the 2-D map of the RhD protein but greatly affected the LW map, further indicating that LW, but not RhD, carries N-linked carbohydrate chains. After deglycosylation the LW map was different from the RhD map, confirming that the RhD and LW polypeptides are different proteins. These findings demonstrate that LW is neither a glycosylated form of Rh protein nor is Rh a precursor of LW.

Properties of the blood group LW glycoprotein and preliminary comparison with Rh proteins.

The major component immunoprecipitated from human red cell membranes by murine monoclonal antibodies (BS46 and BS56) against the LW blood group antigens is a 42,000 mol. wt glycoprotein. Upon digestion by an N-glycanase the LW component migrated as a 25,000 mol. wt component on SDS gels, whereas treatment by an O-glycanase led only to a small size reduction (2000). These data suggest that the LW glycoprotein might carry approximately eight to nine N-linked sugar chains and only a few (one or two) O-linked oligosaccharide chains. A minor component of 31,000 mol. wt was also identified in the LW immunoprecipitate. Preliminary analyses by two-dimensional peptide mapping indicate that the 31,000 mol. wt polypeptide is identical to authentic Rh proteins, therefore raising the possibility that the Rh and LW antigens are associated in the membrane as a functional complex called Rh cluster. Since the N-deglycosylated form of the LW and RhD proteins have different sizes (25,000 vs 31,000-32,000 respectively) and since their externally 125I-labelled domains have different two-dimensional peptide maps, it is concluded that LW is probably not a simple glycosylated form of the Rh proteins.

Two-dimensional iodopeptide mapping demonstrates that erythrocyte Rh D, c, and E polypeptides are structurally homologous but nonidentical.

The 32,000 molecular weight (mol wt) erythrocyte Rh D, c, and E polypeptides were separately purified from cDE/cDE erythrocytes by monoclonal immunoprecipitations and compared by two-dimensional iodopeptide mapping. Digestions of the isolated Rh polypeptides with alpha-chymotrypsin revealed a high degree of structural homology between c and E (13/14 iodopeptides were identical) and less striking homology between D and c or E (8/19 identical). The iodopeptide maps of Rh proteins purified by a nonimmunologic protocol from cDE/cDE erythrocytes were virtually identical to the composite pattern (D + c + E) deduced from the individual maps of Rh D, c, and E iodopeptides. Digestions of the isolated Rh polypeptides with trypsin revealed an overall homology of approximately 50% between iodopeptides derived from D, c, and E. These data indicate that the erythrocyte Rh D, c, and E antigens are carried by homologous but distinct molecular species; c and E appear more closely related to each other than to D.

High frequency antigens of human erythrocyte membrane sialoglycoproteins, V. Characterization of the Gerbich blood group antigens: Ge2 and Ge3.

The molecular properties of the major, high-frequency antigens (Ge2 and Ge3) of the human Gerbich blood group system were investigated using 14 different alloantibodies from rare Ge: -1,-2,-3 or Ge: -1,-2,3 individuals. Various modification, fractionation or fragmentation products of glycophorins (sialoglycoproteins) from normal erythrocytes (phenotype Ge: 1,2,3) were used in hemagglutination inhibition assays. The location of the antigens was also studied by blotting of proteins, separated by dodecyl sulfate polyacrylamide gel electrophoresis, to nitrocellulose and detection of bound antibodies by 125I-labelled protein G. Anti-Ge3 was found to be directed against a region of glycophorin C that surrounds a tryptic cleavage site at position 48 and a similar region of glycophorin D whose structure is not yet known. NeuAc residue(s), probably representing part(s) of a carbohydrate unit attached to serine42 of glycophorin C, methionine, aspartic or glutamic acid, tryptophan and/or arginine residue(s) are involved in the Ge3 epitopes, as judged from chemical modification. The Ge2 epitopes were found to be located on a tryptic glycopeptide from glycophorin D comprising about 20-30 amino-acid residues. NeuAc residue(s), attached to serine-/threonine-linked oligosaccharide(s), are involved in the Ge2 determinants. Using the immunoblotting technique, it could also be shown that the 'new' glycophorin in Ge: -1,-2,3 cells carries the Ge3 antigen.