Analysis of the CC chemokine receptor 3 gene reveals a complex 5' exon organization, a functional role for untranslated exon 1, and a broadly active promoter with eosinophil-selective elements.

To understand the regulation of CC chemokine receptor 3 (CCR3) expression, its gene structure and promoter have been characterized. The CCR3 gene contains 4 exons that give rise to multiple messenger RNA (mRNA) species by alternative splicing. Exon 1 is present in all transcripts, whereas exon 2 or 3 is present at low frequency (< 10%). Exon 4 contains the open reading frame and 11 bp of the 5' untranslated region. Northern analysis revealed 4 species of CCR3 mRNA. Direct sequencing revealed that the first 1 kb of the promoter and exon 1 contained only one mutation in 19 individuals, indicating that the CCR3 promoter and exon 1 are conserved between individuals. The first 1.6 kb of the 5' flanking region of exon 1 contained promoter elements including a TATA box and motifs for myeloid transcription factors and had strong promoter activity in eosinophilic, lymphoid, myeloid, and respiratory epithelial cell lines. Deletion analysis revealed differential regulation of the CCR3 promoter in eosinophilic and epithelial cells suggesting the presence of lineage-specific elements. Interestingly, exon 1 enhanced the activity of the promoter and this effect was especially prominent in eosinophilic cells. Thus, the human CCR3 gene has a complex 5' exon structure, a conserved promoter with strong activity in multiple cell types, and a functional 5' untranslated exon.

Can Dexter cultures support stem cell proliferation?

The in vitro culture of mouse bone marrow (Dexter cultures) has allowed a detailed analysis of the biology of murine hematopoiesis. However, attempts to develop stable long-term human bone marrow cultures have been unsuccessful. Available culture systems all have finite and relatively short lifetimes. The reasons for the limited longevity are unknown. Utilizing computer-assisted integration techniques, we have theoretically simulated culture cell production kinetics to help identify factors that may be responsible for culture decay, as well as to suggest possible means of improving culture longevity. The simulation demonstrates that removal of stem cells is a possible mechanism leading to culture decline. Under the standard bone marrow culture conditions, even with a high stem cell renewal rate, the cultures appear to be destined to fail. Thus, the development of proper sampling techniques or improved stem cell retention may be critical to obtain successful long-term cultures.

Sources of antibody-dependent cellular cytotoxicity deficiency in young pig leukocytes.

Antibody-dependent cellular cytotoxicity (ADCC) activity against pseudorabies virus-infected target cells has been found to be lower in young pig peripheral blood leukocytes (PBLs) than in adults. Experiments were designed to investigate the reason(s) for low activity in the young, which are more at risk of fatal infection than adults. The percentage of polymorphonuclear leukocytes (PMNs), the major ADCC effector cell, in the whole leukocyte population did not have a bearing on the deficiency. Enrichment for PMNs did not alleviate differences in activity between young and adult pigs. Additionally, no suppressor cell(s) or factor(s) could be demonstrated to account for the ADCC deficiency. The source of the ADCC deficiency in the young was found to be related to the decreased ability of young pig effector cells to bind antibody-sensitized targets. This deficiency relative to adults was associated with decreased antibody binding to high affinity Fc receptors on young pig neutrophils.

Lack of formyl-methionyl-leucyl-phenylalanine receptors on porcine neutrophils.

The response of blood neutrophils to the chemotactic peptide formyl-methyl-leucyl-phenylalanine varies among species. Our results indicate that this peptide does not activate the respiratory burst of porcine neutrophils. Specifically, concentrations less than or equal to 10(-6) M did not cause production of either superoxide or hydrogen peroxide. Studies designed to delineate the biochemical deficit responsible for these results indicated that these cells do not express specific chemotactic peptide receptors on the external surface of the plasma membrane. Although these data do not rule out the possibility that internal stores of chemotactic peptide receptor exist, attempts to induce expression of the receptor by priming the cells with either lipopolysaccharide or calcium ionophore were unsuccessful.

Characterization of porcine peripheral blood leukocytes by light-scattering flow cytometry.

As a basis for other experiments using flow cytometry of porcine peripheral blood leukocytes, cell fractions were isolated by various methods and analyzed by forward angle light scatter and 90 degree light scatter. Cytospin smears of cell samples were also studied by leukocyte differential counts and nonspecific esterase staining. Three main populations of peripheral blood leukocytes [lymphocytes, monocytes, and granulocytes (primarily neutrophils)], were defined in the log 90 degree light scatter by forward angle light scatter histogram. Partial overlap was observed between lymphocyte and monocyte, and between monocyte and granulocyte domains. Correlation between leukocyte differential counts and flow cytometric quantification based on bitmap statistics of appropriate domains was between r = 0.872-0.892 for lymphocyte and granulocyte. Percoll density gradients were used for subfractionation of leukocyte populations, especially for the enrichment of granulocytes. The specific densities were calculated for lymphocytes (1.0585-1.0819 g/cc), monocytes (1.0585-1.0702 g/cc), granulocyte (1.0819-1.0936 g/cc), and erythrocytes (greater than 1.0952 g/cc). We suggest that light scatter characterization is a basis for future studies of porcine blood by flow cytometry.

Swine antibody-dependent cellular cytotoxicity against pseudorabies virus-infected cells.

In pseudorabies virus (PRV) infection of pigs, antibody-dependent cellular cytotoxicity (ADCC) may be an early defense mechanism. Peripheral blood leukocytes (PBL) and pulmonary macrophages mediate ADCC activity. Antibody-dependent cellular cytotoxicity against PRV-infected target cells was assessed, and the effect of infection of cells having an ADCC-effector function was determined. Although pulmonary lavage cells (PLC) had ADCC activity, in vitro infection of PLC led to PRV replication, loss of cell viability, and loss of ADCC activity. In contrast, infection of PBL did not lead to replication, decreased cell viability, or reduced ADCC activity, compared with those in non-infected controls. Measuring ADCC activity in a longitudinal study revealed that PBL from neonates had lower ADCC activity than did PBL from pigs greater than 3.5 months old. Peripheral blood leukocytes and not PLC may have a greater role in control of PRV dissemination in the pig. The difference in activity between cells from neonates and older pigs might explain, in part, the age dependency in the severity of the disease.