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Infection and Immunity, June 2001, p. 3678-3684, Vol. 69, No. 6
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.6.3678-3684.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Expression of Cytokine and Chemokine Genes by Human Middle Ear Epithelial Cells Induced by Formalin-Killed Haemophilus influenzae or Its Lipooligosaccharide htrB and rfaD Mutants

H. H. Tong, Y. Chen, M. James, J. Van Deusen, D. B. Welling, and T. F. DeMaria*

Department of Otolaryngology, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio

Received 3 January 2001/Returned for modification 31 January 2001/Accepted 2 March 2001

To define the role of nontypeable Haemophilus influenzae (NTHI) lipooligosaccharide (LOS) in the induction of proinflammatory cytokine gene expression during otitis media, we compared the abilities of formalin-killed NTHI strain 2019 and its LOS htrB and rfaD mutants to stimulate human middle ear epithelial (HMEE) cell cytokine and chemokine gene expression and production in vitro. Strain DK-1, an rfaD gene mutant, expresses a truncated LOS consisting of only three deoxy-D-manno-octulosonic acid residues, a single heptose, and lipid A. Strain B29, an isogenic htrB mutant, possesses an altered oligosaccharide core and an altered lipid A. HMEE cells were incubated with formalin-killed NTHI 2019, B29, or DK-1. The supernatants and the cells were collected at 2, 4, 8, and 24 h after stimulation. Expression of genes for the cytokines tumor necrosis factor alpha (TNF-alpha ), interleukin lbeta (IL-1beta ), and IL-6 and for the chemokines macrophage inflammatory protein 1beta (MIP-1beta ), monocyte chemotactic peptide 1 (MCP-1), and IL-8 was quantitated by real-time PCR. NTHI B29 did not significantly stimulate any cytokine or chemokine mRNA expression in HMEE cells. In striking contrast, NTHI 2019 induced up to 105-, 139-, and 187-fold increases in HMEE cell expression of IL-1beta , TNF-alpha , and MIP-1beta , respectively (P < 0.01 [2019 versus B29]). NTHI 2019 also induced upregulation of IL-8, IL-6, and MCP-1 mRNA expression (by 26-, 44-, and 14-fold, respectively [P < 0.05 {2019 versus B29}]). The significant induction of cytokine genes was confirmed by quantitating the secretion of cytokines in culture supernatants with an enzyme-linked immunosorbent assay. There were no significant differences in mRNA expression of IL-8, IL-6, and MCP-1 between the 2019- and DK-1-treated groups. The low levels of gene transcripts observed after incubation of HMEE cells with B29 indicate that products of the disrupted NTHI htrB LOS gene may play a major role in induction of these particular inflammatory mediators.

* Corresponding author. Mailing address: Department of Otolaryngology, The Ohio State University, Room 4331 UHC, 456 West 10th Ave., Columbus, OH 43210. Phone: (614) 293-8103. Fax: (614) 293-5506. E-mail: demaria.2{at}osu.edu.

Infection and Immunity, June 2001, p. 3678-3684, Vol. 69, No. 6
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.6.3678-3684.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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