CCR6-deficient mice have impaired leukocyte homeostasis and altered contact hypersensitivity and delayed-type hypersensitivity responses.

CCR6 expression in dendritic, T, and B cells suggests that this beta-chemokine receptor may regulate the migration and recruitment of antigen-presenting and immunocompetent cells during inflammatory and immunological responses. Here we demonstrate that CCR6-/- mice have underdeveloped Peyer's patches, in which the myeloid CD11b+ CD11c+ dendritic-cell subset is not present in the subepithelial dome. CCR6-/- mice also have increased numbers in T-cell subpopulations within the intestinal mucosa. In 2,4-dinitrofluorobenzene-induced contact hypersensitivity (CHS) studies, CCR6-/- mice developed more severe and more persistent inflammation than wild-type (WT) animals. Conversely, in a delayed-type hypersensitivity (DTH) model induced with allogeneic splenocytes, CCR6-/- mice developed no inflammatory response. The altered responses seen in the CHS and DTH assays suggest the existence of a defect in the activation and/or migration of the CD4(+) T-cell subsets that downregulate or elicit the inflammation response, respectively. These findings underscore the role of CCR6 in cutaneous and intestinal immunity and the utility of CCR6-/- mice as a model to study pathologies in these tissues. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Expression of CCR9 beta-chemokine receptor is modulated in thymocyte differentiation and is selectively maintained in CD8(+) T cells from secondary lymphoid organs.

Chemokines appear to have an important role in the seeding of lymphoid progenitors in the thymus, the regulation of the coordinated movements of the maturing T cells within this organ, and the egress of the resulting naive T cells to secondary lymphoid organs. CCR9, the specific receptor for the beta-chemokine TECK/CCL25, is selectively expressed in thymus, lymph node, and spleen. Using a specific anti-CCR9 polyclonal antibody, K629, and a semiquantitative reverse transcriptase-polymerase chain reaction procedure, a detailed study of CCR9 expression in the thymus and secondary lymphoid organs was performed. The results show that CD4(+)CD8(+) double-positive thymocytes have the highest CCR9 expression in thymus. Single-positive CD8(+) thymocytes continue to express this receptor after abandoning the thymus as mature naive T cells, as suggested by the existence of a CD8(+)CD69(low)CD62L(high) CCR9(+) cell subset. Consistent with this, CD8(+) lymphocytes from lymph nodes, spleen, and Peyer patches express a functional CCR9, as its expression correlates with migration in response to CCL25. Conversely, CD4(+) thymocytes lose CCR9 before abandoning the thymus, and CD4(+) T cells from secondary lymphoid organs also lack CCR9 expression. Analysis of CCR9 expression in thymocytes from mice of different ages showed that CCR9 levels are affected by age, as this receptor is more abundant, and its response to CCL25 is more potent in newborn animals. Collectively, these results suggest that CCR9 has a role in thymocyte development throughout murine life, with clear differences between the CD4(+) and CD8(+) lineages.

The transient expression of C-C chemokine receptor 8 in thymus identifies a thymocyte subset committed to become CD4+ single-positive T cells.

Developing T cells journey through the different thymic microenvironments while receiving signals that eventually will allow some of them to become mature naive T cells exported to the periphery. This maturation can be visualized by the phenotype of the developing cells. CCR8 is a ss-chemokine receptor preferentially expressed in the thymus. We have developed 8F4, an anti-mouse CCR8 mAb that is able to neutralize the ligand-induced activation of CCR8, and used it to characterize the CCR8 protein expression in the different thymocyte subsets. Taking into account the intrathymic lineage relationships, our data showed that CCR8 expression in thymus followed two transient waves along T cell maturation. The first one took place in CD4(-) CD8(-) double-negative thymocytes, which showed a low CCR8 expression, and the second wave occurred after TCR activation by the Ag-dependent positive selection in CD4(+) CD8(+) double-positive cells. From that maturation stage, CCR8 expression gradually increased as the CD4(+) cell differentiation proceeded, reaching a maximum at the CD4(+) CD8(-) single-positive stage. These CD4(+) cells expressing CCR8 were also CD69(high) CD62L(low) thymocytes, suggesting that they still needed to undergo some differentiation step before becoming functionally competent naive T cells ready to be exported from the thymus. Interestingly, no significant amounts of CCR8 protein were detectable in CD4(-) CD8(+) thymocytes. Our data showing a clear regulation of the CCR8 protein in thymus suggest a relevant role for CCR8 in this lymphoid organ, and identify CCR8 as a possible marker of thymocyte subsets recently committed to the CD4(+) lineage.

Down-regulation of the beta-chemokine receptor CCR6 in dendritic cells mediated by TNF-alpha and IL-4.

Chemokines are involved in the control of dendritic cell (DC) trafficking, which is critical for the immune response. We have generated DC from human umbilical cord blood CD34+ progenitors cultured with granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha (TNF-alpha), and stem cell factor. Using an anti-CCR6 monoclonal antibody, we observed that these cells showed maximum expression of this beta-chemokine receptor when they were immature, as determined by their relatively low expression of several DC maturation markers such as CD1a, CD11c, CD14, CD40, CD80, and CD83. Immature DC responded strongly to macrophage inflammatory protein-3alpha (MIP-3alpha), the CCR6 ligand, in migration and calcium mobilization assays. CCR6 expression decreased in parallel with the DC maturation induced by prolonged TNF-alphaq treatments. Interleukin-4 was also able to decrease CCR6 protein levels. Our findings suggest that the MIP-3alpha/CCR6 interaction plays an important role in the trafficking of immature DC to chemokine production sites such as injured or inflamed peripheral tissues, where DC undergo maturation on contact with antigens.

SA-1, a nuclear protein encoded by one member of a novel gene family: molecular cloning and detection in hemopoietic organs.

We report the molecular cloning of a novel gene family. The first member of this family was cloned from a mouse lambda gt11 cDNA library using the B92 monoclonal antibody (mAb) raised against stromal cell extracts. This was followed by RACE-PCR using mRNA from the stromal cell line. A 4 kb cDNA was obtained encoding a unique protein sequence of 1258 aa, that we designate stromal antigen (SA)-1. The human SA-1 gene was cloned by homology from a thymus cDNA library and the sequence of the predicted protein was found to be highly homologous to the murine SA-1 (>98.9%). Another cDNA was cloned and the deduced protein (SA-2) was 71% homologous to SA-1. Northern blot and PCR analysis indicated that on the mRNA level the SA-1 gene is expressed in all tissues analyzed and probably encodes a single transcript. The identification of SA-1 protein in tissues and cells required combined immunoprecipitation and Western blotting using a polyclonal antiserum raised against a predicted peptide of SA-1 and the B92 mAb. Using this assay we identified a protein of about 120 kDa in hemopoietic organs. Subcellular fractionation indicated that SA-1 is a nuclear protein. Thus, despite the ubiquitous expression on the mRNA level, the protein was predominantly detected in hemopoietic organs and may therefore be controlled on a post-transcriptional level. The SA-1 gene described in this study is highly conserved between mouse and man. This implies a crucial function for this protein.

zhx-1: a novel mouse homeodomain protein containing two zinc-fingers and five homeodomains.

A cDNA encoding the complete reading frame of a novel homeodomain-containing protein, named zhx-1, has been cloned from a mouse bone marrow stromal cell line. The ORF encodes a protein of 873 amino acids, in which two C2-H2 zinc-fingers and five homeodomains have been identified. These features classify zhx-1 as a member of the ZF class of homeodomain transcription factors, zhx-1 mRNA is widely expressed in adult mouse, although it is preferentially expressed in brain. At least, two homologous mRNAs are present in humans suggesting that zhx-1 is the first member of a novel subclass of homeodomain factors.

Transformation of Penicillium chrysogenum to sulfonamide resistance.

Penicillium chrysogenum has been transformed to sulfonamide resistance by vectors containing the dihydropteroate synthetase gene from plasmid R388 controlled by the promoter and terminator sequences of the P. chrysogenum trpC gene. Transformation frequencies of four to ten transformants per microgram of vector DNA were obtained.

Cloning and characterization of the isopenicillin N synthetase gene mediating the formation of the beta-lactam ring in Aspergillus nidulans.

Genomic clones containing an Aspergillus nidulans isopenicillin N synthetase (IPNS) gene have been identified by heterologous hybridization with a Cephalosporium acremonium DNA probe. The open reading frame encodes a 331 amino acid polypeptide with extensive homology with the genes of other beta-lactam-producing fungi. The gene product has been overexpressed in Escherichia coli and shown to have activity of IPNS. This represents the first evidence at the molecular level that the biosynthesis of penicillins in A. nidulans occurs by the same pathway as in other beta-lactam-producing microorganisms. Comparison of available nucleotide sequences from IPNS genes suggests a horizontal transmission of the gene between the prokaryotic beta-lactam producers of the genus Streptomyces and the filamentous fungi.

Excess function hairy-wing mutations caused by gypsy and copia insertions within structural genes of the achaete-scute locus of Drosophila.

Hairy-wing (Hw) mutations cause the differentiation of extra chaetes on the cuticle of Drosophila. They are associated with modifications of the achaete-scute complex that consist, in the mutants studied, of insertions of the transposable elements gypsy (Hw1, HwBS) or copia (HwUa). gypsy and copia are inserted in achaete and scute transcribed regions, respectively. Transcription of the insertion-split genes starts at the normal site but terminates within the transposable element sequences. The RNA truncated within gypsy is 5-20 times more abundant than its homolog in wild-type flies. The abundance is reduced in Hw1 revertants and Hw1 stocks carrying su(Hw) mutations. These and other data suggest that the excess function phenotypes of Hw mutations are generated by an increase in achaete or scute transcripts.

Molecular genetics of the achaete-scute gene complex of D. melanogaster.

The achaete-scute gene complex (AS-C), involved in differentiation of the sensory chaetes of D. melanogaster, and the yellow locus have been cloned. The yellow locus is the most distal and is followed, proximally, by the achaete and the scute loci. In the scute locus (75 kb), three transcription units separated by long stretches of DNA give rise to poly(A)+ RNAs of 1.6, 1.2, and 1.6 kb. Most DNA lesions associated with scute mutations map within the presumably untranscribed DNA. Their mutant phenotypes are stronger the closer the lesions are to the structural gene of one transcript (T4 RNA). Genetic and developmental data suggest that only this RNA is fundamental for the scute function. Its transcription might be perturbed by far removed DNA lesions. A second transcript is probably implicated in the lethal of scute embryonic function, while the third transcript is unnecessary for the differentiation of most macrochaetes. Two additional polyadenylated RNAs are transcribed from the achaete (1.1 kb) and yellow (1.9 kb) loci.

DNA map of mutations at the scute locus of Drosophila melanogaster.

The achaete-scute gene complex (AS-C) of Drosophila melanogaster is involved in the differentiation of innervated elements in the adult (chaetes) and in the embryo (central nervous system). Genetically, the AS-C is subdivided into four regions: achaete, scute alpha, lethal of scute, and scute beta. Using a previously cloned fragment of scute DNA, we have now cloned 62 kb of wild-type DNA from the scute region. No repetitive sequences have been detected in this stretch of DNA. Of 16 scute mutants with chromosomal rearrangements studied (inversions, deletions, and translocations), nine, included genetically in scute beta, have breakpoints in the cloned region. The remaining rearrangements, which genetically correspond to scute alpha, map outside and to the left of the cloned region. Of nine scute ;point mutants' studied, eight have large DNA alterations within the cloned region. These alterations include insertions (five) and deletions (three). The DNA alterations found in both ;point mutants' and rearrangements are interspersed and scattered over 40 kb. The relationship between the sites of the DNA alterations and the mutant phenotypes are discussed.