Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein

We examined the bactericidal activity of two proteins that are abundant in the cytoplasmic granules of human eosinophils, major basic protein (MBP) and eosinophil cationic protein (ECP). Unlike the human neutrophil's peptide defensins, both MBP and ECP killed stationary phase Staphylococcus aureus 502A in a simple nutrient-free buffer solution. Although MBP also killed Escherichia coli ML-35 with considerable efficacy under these experimental conditions, the in vitro activity of ECP against E. coli was considerably enhanced if mid-logarithmic phase bacteria replaced stationary phase organisms or if the assay medium was enriched with trypticase soy broth. The antibacterial activity of both eosinophil proteins was modulated by incubation time, protein concentration, temperature and pH. A pBR322-transformed derivative of E. coli ML-35 was used to examine the effects of ECP and MBP on integrity of the bacterial inner membrane (IM) and outer membrane. Although both MBP and ECP caused outer and inner membrane permeabilization when nutrients were present, only MBP was effective under nutrient-free conditions. Two proton ionophores (DNP and carbonyl cyanide m-chlorophenyl hydrazone) protected E. coli from the bactericidal effects of ECP but not from MBP. These findings establish that MBP and ECP have bactericidal properties and suggest that these proteins kill E. coli by similar but nonidentical mechanisms marked by an attack on the target cell's membranes. In view of evidence that high concentrations of ECP and MBP exist in cytoplasmic granules whose contents are translocated to phagocytic vacuoles, we suggest that MBP and ECP contribute to the eosinophil's ability to kill ingested bacteria.     

The complement factor 5a receptor gene maps to murine chromosome 7

Complement factor 5a (C5a) promotes local inflammation and is a potent chemoattractant for neutrophils and macrophages. We had an interest in C5a and its receptor, C5r1, because we previously identified C5a as a positional candidate gene for the quantitative trait locus Abhr2, which determines allergen-induced bronchial hyperresponsiveness in our murine model of asthma. To study the significance of C5r1 in our asthma model we first had to determine its genomic map location in mice. Genomic sequence surrounding murine C5r1 was analyzed for polymorphisms and two variable microsatellites were identified. These microsatellites were genotyped in A/J x (C3H/HeJ x A/J)F1 backcross mice (n = 355) and mapped in a panel of 164 markers spaced at approximately 10 cM intervals throughout the genome. Multipoint linkage analysis placed C5r1 on murine chromosome 7, 3.9 cM from the top of the linkage group. This map location has been previously identified as containing an additional quantitative trait locus for allergen-induced airway hyperresponsiveness, Abhr3, in this population of mice.     

The murine Il-6 gene maps to the proximal region of chromosome 5

Murine Il-6 cDNAs were isolated by cross-hybridization with a human IL-6 cDNA from an IL-1 activated bone marrow stromal cell line (W20). Mouse-hamster somatic cell hybrids were utilized to localize murine Il-6 to chromosome 5. Genetic mapping with respect to En-2, AlbH, and Gus in backcross progeny from an interspecific mating between C57BL/6J and Mus spretus positioned Il-6 3 cM distal to En-2. The syntenic relationships of Il-6 and En-2 in mouse and man, as well as the potential role of IL-6 in tumorigenesis, are discussed.     

The structural gene for F liver protein (Flp) maps to chromosome 5 of the mouse

The BXD and AKXL panels of recombinant inbred mouse strains have been typed for the F liver protein alloantigen. The structural gene for F liver protein gene (Flp) is placed on the distal part of chromosome 5, between the known markers Bcd-1 and Gus-s. This excludes the possibility that F liver protein is a major histocompatibility complex molecule, and in turn raises a question about the uniqueness of F and certain other proteins as purgers of self-reactivity among T but not B cells. The typed RI strains have then been used for the immunogenetic studies presented in the succeeding article.     

The gene for T11 (CD2) maps to chromosome 1 in humans and to chromosome 3 in mice

The chromosomal locations of the human and murine T11 (CD2) gene have been determined. Using recently cloned cDNA to probe Southern blots of mouse X human and Chinese hamster X mouse somatic cell hybrids, we have localized the human T11 gene to chromosome 1 and the murine T11 gene to chromosome 3. Based on previously determined blocks of homology between human chromosome 1 and mouse chromosome 3, it is suggested that the human T11 gene may lie on the short arm of chromosome 1 proximal to p221. Thus, the T11 gene is not linked to any other genes for T cell markers that have been mapped to date.     

The human alpha 2(XI) collagen gene (COL11A2) maps to the centromeric border of the major histocompatibility complex on chromosome 6

Type XI collagen, a minor structural component of cartilage fibrils, is composed of three chains, alpha 1(XI), alpha 2(XI), and alpha 3(XI). Using a cloned fragment of the human alpha 2(XI) collagen gene (COL11A2) as a molecular probe for in situ hybridization and somatic cell hybrid mapping, we have localized the gene to the short arm of chromosome 6, region 21.3. By exploiting the rich source of probes provided by the major histocompatibility complex (MHC) genes, which also map to this chromosomal band, we have constructed macrorestriction maps of the region by pulsed-field gel electrophoresis and have localized the alpha 2(XI) collagen gene to the centromeric extreme of the MHC. Finally, we have demonstrated, by the isolation of overlapping cosmid clones, that the gene is 45 kb centromeric to the HLA-DPB2 locus and oriented with the 3' end toward the MHC. The COL11A2 locus thus demarcates the proximal boundary of the MHC. This finding may have implications for the understanding of certain MHC-linked diseases.