Experimental Studies On Tracheal Epithelial Cells In Vitro By Nontypeable Haemophilus Influenzae And The Effect Of Bacterial Infection On Airway Inflammation Of Acute Exacerbation Of Chronic Obstructive Pulmonary Disease

Obstrucive To explore the interaction of nontypeable Haemophilus influenzae (NTHi) strain ATCC49247 with primary rabbit tracheal epithelial (TE) cells; to study the interaction of NTHi with primary human bronchial epithelial cells (HBEC) and the NTHi-induced release and expression of proinflammoraty cytokines in the HBEC; to investigate the respiratory infection of outpatients with acute exacerbations of COPD (AECB) and to evaluate the relationship among sputum color, white cell count and bacterial positivity; to assess the possible relationship between airway inflammation and airway bacterial infection of acute exacerbations of chronic obstructive pulmonary disease(AECB) by bacterial infection.Methods (1) TE cells were isolated by using low temperature protease digestion. TE cells were cultured on collagen gel-coated membranes at an air-liquid interface in serum-free medium. Under these conditions,TE cells first proliferated and then differentiated into a pseudostratified mucociliary epithelium. Then, TE cells were infected by NTHi. After 24h, fixed cell cultures were processed for Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM); (2) HBEC were isolated by using low temperature protease digestion and cultured in serum-free medium. Confluent epithelial cell were incubated with NTHi, NTHi +erythromycin (0.lmg/L) , NTHi + erythromycin (l0mg/L), NTHi + gentamicinC l00mg/L), NTHi+dexamethasone(0.1mM) and normal untreated control cells after 24h, respectively. Release of interleukin(IL)-8 and tumor necrosis factor(TNF)- α into the supernatant was assayed by enzyme-linked immunosorbent assay. The expression of ICAM-1 was examined by immunohistochemistry staining; (3) Samples of satisfactory sputum from 159 patients with AECB were collected and cultured. The macroscopic appearance of the samples was allocated a sputum number. Total and differential cell counts were examined. Relationship between sputum color and bacterial positivity was studied; (4) Ninety-seven spontaneous sputum samples obtained from 97 patients with AECB were classfied into five groups, which Pseudomonas aeruginosa, Klebsiella penumoniae bacilli, Haemophilus influenzae(Hi) and Haemophilus parainfluenzae(HP) were isolated as sole pathogens andonly normal florae were isolated. Induced sputum samples were obtained from 15 healthy persons. Total and differential cell counts were examined. Levels of sputum IL-8, interleukin(IL)-6 and TNF-a were measured by enzyme-linked immunosorbent assay(ELISA).Results (1) SEM indicated that bacteria adhered to nonciliated cells in the population. 90 percent of TE cells were either died or apotosised and cilia appeared to be broken. TEM showed lamellipodia and microvilli surrounding organisms, the presence of bacteria within vacuoles of airway cells and organisms adhering to the cell surface. (2) NTHi-induced cells released significant quantities of IL-8 and TNF and increased the total number of ICAM-1 positive cells compared to normal untreated cells (P<0.001). Incubation of HBEC with 0.lmg/L erythromycin and gentamicin stilll significantly induced the release of IL-8, TNF-a and the expression of ICAM-1, which was blocked by l0mg/L erythromycin and 0.lmM dexamethasone. Similar to observations with A549 cells ,infection with NTHi resulted in higher levels of IL-8, TNF-oc and ICAM-1, which was blocked by l0mg/L erythromycin. (3) Ninety-two strains of pathogens were isolated (52.9%). Major pathogens included Haemophilus influenzae, Klebsiella penumoniae bacilli and Pseudomonas aeruginosa. There were 57 srains of Haemophilus parainfluenzae from 159 sputums. Bacterial positivity was 82.7% from 87 samples of purulent sputum and 27.8% from 72 samples of mucoid sputum (P<0.001). Total white cell counts were increased in purulent sputum compared to mucoid sputum (P<0.001). (4) Levels of sputum IL-8, IL-6, TNF- and neutrophil counts in the AECB were significantly higher than those in the healthy persons. Pseudomonas aeruginosa, Klebsiella penumoniae bacilli and Hi exacerbations we...