[Advances of studies on the occurrence of the upper respiratory disease correlative with immunity and tobacco].

SummaryTobacco smoke exposure has obvious and complex effects on the immune system of the human upper respiratory tract, including pro-inflammatory and anti-immune effects. Exposure to tobacco smoke is closely related to the occurrence and development of allergic rhinitis, the common rhinitis and sinusitis. The innate immune system is influenced by tobacco smoking through its effects on the respiratory mucosa and its adjuncts, natural killer cells, dendritic cells, neutrophils and innate immune receptors. Cigarette smoke can also affect the humoral immunity and cellular immunity, altering the acquired immune condition of the upper respiratory tract. Tobacco smoke exposure promotes the occurrence and development of the upper respiratory tract infectious diseases and allergic diseases by changing the composition of microflora in the upper respiratory tract.

Genetic identity and differential expression of p38.5 (Haymaker) in human malignant and nonmalignant cells.

Previous studies from our laboratory revealed a novel protein of 38.5 kD on the surface of malignant cell lines of hematopoetic origin that exhibit susceptibility to naive natural killer (NK) cell-mediated lysis. In contrast, p38.5 was not detected on the surface of NK cell-resistant carcinoma cell lines or normal cells. We now report that this protein is differentially expressed, intracellularly, in malignant cell lines of both hematopoetic and epithelial origin compared with nonmalignant cells. To characterize p38.5 further, we used a previously developed antipeptide antibody (anti-11-mer) to probe cDNA expression libraries and subsequently performed 5' extension by rapid amplification of cDNA ends (RACE). Sequence analyses of these cDNA clones reveal open reading frames (ORFs) that include the previously identified 11-mer peptide from purified, native p38.5 and that have identical sequences to a gene of unknown function on chromosome 19. Nucleotide sequence data obtained from these cDNA clones, as well as analysis of the genomic sequence, permitted design of primers for reverse transcriptase-polymerase chain reaction (RT-PCR) that resulted in a cDNA clone encoding an ORF of 361 amino acids; the clone was identical to a sequence encoded by an unpublished mRNA in GenBank. Anti-p38.5 antibody against the 11-mer peptide encoded in exon 5 and against a 25-mer peptide encoded in exon 1 both reacted with the same protein in immunoprecipitation studies, providing further evidence of identity. RT-PCR and Northern blot analyses both demonstrated p38.5 gene transcripts in normal cells, nonmalignant cell lines and malignant cell lines of epithelial as well as hematopoietic origin. Semiquantitative studies revealed a greater level of p38.5 gene transcription in malignant cell lines compared with nonmalignant cells. Immunoblot analyses of protein expression confirmed and extended the latter studies by revealing substantially greater levels of the 38.5 kD protein in whole cell extracts of malignant cell lines compared with nonmalignant cells. Quantitative differences in detection of the 38.5 kD protein and mRNA in NK susceptible- hematopoietic malignancies compared with NK resistant-carcinomas were not observed. These experiments suggest that the p38.5 gene (Haymaker) is widely expressed in human cells of different tissue origins but that elevated expression is associated with the malignant phenotype.

Preferential interaction of a novel tumor surface protein (p38.5) with naive natural killer cells.

A receptor-ligand interaction exclusive to natural killer (NK) cell-mediated recognition and triggering of tumor cell destruction has not yet been identified. In contrast, molecules that are involved in cellular adhesion and regulation of NK cytolysis have been well studied. In this report, a novel tumor surface protein is identified that exhibits characteristics of a recognition structure for naive NK cells. A tagged ligand-cell adsorption technique revealed a 38.5-kD plasma membrane protein (p38.5) from a prototypical NK-susceptible cell line (K562) that preferentially bound to NK cells (CD3(-)CD5(-)CD16(+)) relative to T lymphocytes (CD3(+)CD5(+) CD16(-)). The molecule was purified to apparent homogeneity for further characterization. An amino acid sequence of an 11-mer internal peptide of p38.5 did not exhibit homology to known proteins. Affinity-purified antibody generated against this peptide (anti-p38.5) reacted with a single protein of 38.5 kD on Western blots of whole cell extracts of K562. Flow cytometry and immunoprecipitation studies of surface-labeled tumor cells demonstrated expression of p38.5 on NK-susceptible tumor cell lines (K562, MOLT-4, Jurkat), whereas p38.5 was not detected on NK-resistant tumor cell lines (A549, Raji, MDA-MB-231). Significantly, p38.5 loss variants derived from wild-type Jurkat and Molt-4 cell lines exhibited decreased susceptibility to NK cell-mediated lysis demonstrating a strong association between cell surface expression of p38.5 and cytotoxicity. Purified p38.5 retained preferential binding to NK cells and inhibited NK activity in a dose-dependent manner, thereby providing direct evidence of a role in the lytic process. Binding studies identified a 70-kD membrane protein from NK cells as a possible receptor for the p38.5 tumor ligand. Consistent with cellular adsorption studies, the 70-kD, p38.5 binding protein was not detected on T lymphocytes. Based on studies demonstrating selective binding of p38.5 to NK cells, lack of expression on NK-resistant tumor cell lines and ability of the purified molecule to block cytolysis, we conclude that p38.5 may serve as a recognition/triggering ligand for naive human NK cells.

Relationship between cellular accumulation of rhodamine 123 (R123) and cytotoxicity in B16 melanoma cells.

Rhodamine 123 (R123) is a mitochondria-specific prototype anticancer agent because its target is the energy-producing mechanism of the cell. The goal of this study was to investigate the relationship between intracellular R123 accumulation and cytotoxicity in a R123-sensitive cell line (RS) and a R123-resistant subline (RR) that we developed. Cytotoxicity after exposure to R123 (0-60 micrograms/ml) was assessed using the clonogenic assay. Intracellular R123 was extracted with acid-alcohol and measured by fluorimetry. The rate of R123 accumulation over 1 hr was significantly higher (P less than 0.0001) for RS cells (4.65 +/- 0.39 micrograms/min/10(6) cells) than for RR cells (1.29 +/- 0.24 micrograms/min/10(6) cells). R123 accumulation in RS cells was strongly correlated (r = 0.80; P less than 0.0001) with cytotoxicity. Treatment of RR cells with verapamil (100 microM) reversed R123 resistance. The resulting dose-survival curve was identical to the dose-response curve of RS cells treated with R123 alone. Cellular content of R123 in RR cells treated with verapamil increased to a level similar to that of RS cells and correlated with cytotoxicity. These data suggest that cytotoxicity of R123 in B16 cells results from increased cellular accumulation of R123.