Charles G. Garlisi

Human ADAM33: protein maturation and localization

ADAM33 (a disintegrin and metalloprotease) was recently found to be a novel asthma susceptibility gene. Domain-specific antibodies were used to study its expression and processing. When the pro-domain and catalytic domain were expressed by a stable-transfected cell line, the pro-domain was removed by cleavage within a putative furin cleavage site. The catalytic domain was active in an alpha(2)-macroglobulin complex formation assay and mutation of the catalytic site glutamic acid (E346A) eliminated activity. In transient transfections using the full-length protein, a pro-form and mature form were detectable and alternate glycosylation was demonstrated at sites within the catalytic domain. ADAM33 was detected on the cell surface, with the majority of protein detected intracellularly. The E346A mutation had no significant effect on protein processing. Endogenous ADAM33 was detected in bronchus tissue, bronchial smooth muscle cells, and MRC-5 fibroblasts, consistent with a role in the pathophysiology of asthma.

Cytokine gene expression in mice undergoing chronic graft-versus-host disease☆

Chronic graft-versus-host disease (GVHD) can be induced in B6D2F1 mice by injection of parental DBA/2 lymphoid cells. Stimulation of donor T cells by host MHC antigens leads to the stimulation of host B cells. Little is known of the lymphokines produced during such a reaction. This study was designed to directly measure the levels of mRNA for interferon-gamma (IFN-gamma), interleukin 2 (IL-2), IL-4, IL-5, and IL-10, as well as several other genes, using semiquantitative polymerase chain reaction (PCR). Semiquantitative PCR was reproducible and signals generated were dependent on the amount of specific RNA or cDNA in each reaction. Early during the progression of GVHD (2 days after the first injection of parental cells) there was little increase in IL-10 mRNA, a slight increase in IL-4 mRNA, and a dramatic increase in IL-2 mRNA. In addition, IL-2 bioactivity was demonstrated in supernatants from GVH splenocytes cultured in vitro for 24 h. Later in the response (1 week after the second and final injection of parental cells) IL-4 mRNA levels were elevated as they were earlier while IL-10 mRNA levels were dramatically increased. IL-2 mRNA levels were no different in mice undergoing GVHD than in normal mice at this time. IFN-gamma mRNA was detectable both early and late, although at similar levels in normal mice and mice undergoing GVHD. At both times examined, IL-4 was below the limits of detection by bioassay and IFN-gamma, IL-4, IL-5 and IL-10 were below the limits of detection by ELISA. Further studies showed that a majority of the IL-4 and IL-10 mRNA found elevated in GVH mice were produced by Thy1.2+ T cells, with small amounts from B220+ B cells. In addition, the detectable IFN-gamma mRNA found in GVH mice at this later time also was produced by Thy1.2+ T cells, with small amounts from B220+ B cells.

The assignment of chemokine-chemokine receptor pairs: TARC and MIP-1β are not ligands for human CC-chemokine receptor 8

Identification of chemokine receptors and their associated ligands is crucial to the understanding of most immune reactions. Three human chemokines [I‐309, thymus and activation‐regulated chemokine (TARC) and macrophage inflammatory protein‐1β (MIP‐1β)] have been reported to be ligands for CC‐chemokine receptor 8 (CCR8). In this report, we present evidence that TARC and MIP‐1β did not bind to or induce chemotaxis through CCR8 on a stable transfected cell line (1D‐21) and did not bind to CCR8 on in vitro differentiated human CD4+ Th2 cell cultures. Also, I‐309‐dependent calcium mobilization in 1D‐21 cells and in Th2 cells was desensitized by I‐309 but not by MIP‐1β or TARC. These results provide strong evidence that, at physiologically relevant concentrations, I‐309 is the only known human ligand for CCR8. These data also provide a framework for suggesting minimum requirements for the assignment of chemokine receptor‐ligand pairs.

Receptor reserve analysis of the human CCR3 receptor in eosinophils and CCR3-transfected cells.

A novel pharmacological study of CCR3 receptor reserve in a CCR3‐transfected cell (CREM3) and human eosinophils was done; functional responses measured were increases in intracellular calcium and chemotaxis. Eotaxin, eotaxin‐2, monocyte chemoattractant protein‐4 (MCP‐4), RANTES, and MCP‐3 induced similar maximal eosinophil chemotaxis, whereas MCP‐3 and RANTES induced submaximal calcium responses in eosinophils compared to eotaxin, MCP‐4, and eotaxin‐2. This suggested a receptor reserve in the chemotaxis response. Receptor reserve was quantitated for eotaxin. Occupancy of all CCR3 receptors was required for a maximal calcium response in both CREM3 and eosinophils (reserve = 1.0 or 0.17, respectively); the stimulus‐calcium response relationship was linear, indicating no receptor reserve. In contrast, in eosinophils a large receptor reserve (6.5) was found for chemotaxis, where occupancy of 15% receptors drove half‐maximal responses. These studies indicate that CCR3 interacts with G‐proteins that are poorly coupled to the calcium response, whereas coupling efficiency and/or amplification to the chemotaxis apparatus in human eosinophils is significantly greater. J. Leukoc. Biol. 67: 441–447; 2000.