230 Bcl6 transcription factor is a novel respressor of Il-5 gene expession

S76

Abstracts

J ALLERGY CLIN IMMUNOL JANUARY 2000

230 Be16 Transcription

Factor

Is a Novel

Repressor

of IL-5

Gene

Expression daf,

M Arima *# H Toyama *, S Okada *, M Hatano *. T FukuT Tokuhisa* *Department of Developmental Genetics. Chiba

University Graduate School of Medicine, Chiba, Japan tDepartment of Allergy and Clinical Immunology, Dokkyo University School of Medicine, Chiba, Japan The BCL6 gene has been identified from the chromosomal tom&cation breakpoint in B cell lymphomas and functions as a sequence-specific transcriptional repressor. The Bc16 gene is ubiquitously expressed in adult murine tissues. Bcl6-deficient (Bcl6-/-) mice displayed massive Th2 type inflammation with eosinophilic infiltration in various tissues, especially heart and lung, after 4-6 weeks of age. When the function of Bc16 in Th2 lymphokine production was examined by stimulating spleen cells from young Bc16-/mice without eosinophilic inflammation with anti-CD3 antibody, IL5 production by Bc16-/- splenocytes was strikingly enhanced to 20-30 fold more than that by Bc16+/+ mice. However, IL-4 production by Bc16-/- mice was around 3 fold greater than that of Bc16+/+ mice, suggesting that expression of the IL-5 gene is directly regulated by Bc16. We found the Bcl6-binding sequence on the 3?fUT of IL-5 cDNA and the binding of Bc16 to the sequence was confirmed by gel retardation assay. Repressor activity of Bc16 on expression of the IL-5 gene was further examined by cotransfection of the Bcl6 expression vector and the IL-5 gene with deletion into NIH3T3 cells. When various doses of the Bc16 expression vector were cotransfected with the full length of IL-5 cDNA, IL-5 production was significantly reduced to 30% of the maximum in a dose dependent manner. In contrast, there were no significant changes in IL-5 productivity by cotransfection of the IL-5 gene deleted with the binding sequence with any doses of Bc16 expression vector examined. These results indicate that Bc16 binds to the 3?fUT of IL-5 gene to repress its expression. This is the first report to demonstrate a silencer region on the IL-5 gene.

231 Distinct Crosby,

Roles of IL-4 S Farme<

and IL-5

M Borchers,

in Respiratory K Larson.

Inflammation

I Brenneise.

Jefl

J Lee. N Lee

Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ We have developed an IL-5 transgenic model of asthma in mice (line NJ.1726) that reproduces many of the histological and pathophysiological changes associated with asthma in the absence of antigenie stimuli. IL-5 expression in these mice occurs from the airway epithelium by using the Clara cell specific promoter CCIO. To assess the contribution of IL-4 and IL5 as inflammatory signals in type Ihypersensitivity reactions occurring in the lung (i.e., allergic pulmonary inflammation), NJ. 1726 mice or NJ. 1726 mice on an IL-4-/background, were sensitized/aerosol challenged with ovalbumin (OVA). The characterization of the OVA sensitization and aerosolchallenged mice include specific assessments of pulmonary gene expression, leukocytic infiltration, histopathologic perturbations. and pathophysiologic changes of lung function. OVA-treated NJ. 1726 mice, relative to wild-type mice, displayed exaggerated histopathologic changes in response to OVA provocation. For example. in comparison to OVA-treated wild-type mice, OVA-treated NJ. 1726 mice displayed higher levels of mucus production (Mucus index = IO.4 vs. 17.6). and increased eosinophil infiltration of the airway lumen (0.034 vs. 3.4 X 10-e). However, OVA-treatment of NJ. 1726 mice did not result in a further increase in AHR relative to the 2-3 fold increase inherent (i.e., in the absence of allergen challenge) with this transgenie line. We have begun the process of identifying signals and/or cell types that contribute to the OVA phenotype in an IL-5 independent fashion using this nansgenic model. In comparison to OVAtreated NJ. 1726 mice, transgenic animals on an IL-4-/- background display a reduction of eosinophil influx into the lung and BAL as well as dramatically reduced goblet cell numbers and mucus production. Although deletion of IL-4 appears to attenuate many of the exaggerated OVA responses in NJ.1726, AHR is unaffected in the absence of IL-4. Moreover, deletion of IL-4 also has no effect on the intrinsic

(i.e., antigen independent) AHR found in this transgenic line of mice, suggesting that this pathophysiologic change is mediated by mechanisms that are independent of the histopathologic changes occurring in these mice.

232 Prevention

of Airways Eosinophilia in Mice Receiving SubseSensitization and in Mice With Ongoing IgE Responses CpG Oligodeoxynucleotide Zhikang Peng, Xiaojuan Mao, Estelle Simons University of Manitoba, Winnipeg, Canada Increased IgE and airways eosinophila resulting from a predominant Th2 response are important hallmarks of asthma. Unmethylated CpG motifs (CpG) in bacterial DNA or synthesized oligodeoxynucleotides (ODN) are gaining recognition as potential adjuvants selectively inducing a Thl predominant response. We investigated the effect of CpG ODN vaccination on IgE responses and airways eosinopilia in a mouse model of asthma in which mice were sensitized by injections of ovalbumin (OVA) without adjuvant. Groups of BALB/c mice were sensitized intradermally (id.) with OVA (10 ug) twice weekly starting at week 0 for 7 weeks. CpG ODN (30 ug) alone was injected id. one week before sensitization (group A) or a mixture of OVA and CpG ODN was injected i.d. at weeks 0 (B). I (C), and 2 (D), respectively. Airways eosinophilia was induced I week after the last i.d. sensitization by intranasal (i.n.) challenge of OVA (50 ug) once a day for three days. One group of the OVA-sensitized mice received i.n. administration of CpG ODN 2 days before nasal challenge (E), and another group of the sensitized mice received i.n. administration of a mixture of OVA and CpG ODN on the first day of nasal challenge (F). Two days later, after the last challenge, the mice were sacrificed; bronchoalveolar lavage fluids were obtained and eosinophils were counted; OVA-induced spleen cell cytokine productions were performed. Blood samples were obtained bi-weekly and serum OVA-specific IgE and IgG2a antibodies and IL- I2 levels were measured by ELISA. Mice receiving OVA injections and mice receiving saline injections served as positive (G) and negative (H) controls. respecnvely. Systemic administration of CpG ODN significantly inhibited subsequent IgE responses (p’s < 0.05) and airways eosinophilia (p’s < 0.002) by downregulating Th2 and upregulating Thl responses. This effect was found only when CpG ODN was administered one week before or at the time that sensitization started (A & B). Once sensitization commenced, systemic administration of CpG ODN failed to downregulate the ongoing IgE responses and subsequent airways eosinophilia C & D), although serum lgG2a levels stayed high and serum IL- I2 levels increased as did as group A. However, in the sensitized mice, when CpG ODN was administered intranasally before or on the first day of nasal challenge, airways eosinophilia was even more significantly suppressed (E & F) (means = 17% and 9%, p’s < 0.0002) than the systemic administration of the vaccine (A & B) (means = 37% and 24%. p’s < 0.002). although serum IgE levels stayed high. CpG ODN vaccination is more effective when administered systemically on the first day of the sensitization (B 24%) or administered intranasally on the first day of the nasal challenge (F 9%) than when administered before sensitization (A 37%) or challenge (E 17%). We conclude that in this naturally sensitized model, CpG ODN vaccination is a potentially useful approach for preventing airways eosinophilia, even in mice with ongoing IgE responses. quent Using

233 ISS Conjugated Switch

in PBMC

shall+. steinf,

JJ Eiden*,

to Amb From

a 1 Allergen Promotes a Th2 to Thl Ragweed-Allergic Humans JD Mar-

A Kagqv-Sobotkat,

PS Creticosf.

LM Lichten-

G Van Nesr* *Dynavax Technologies Corporation, Berkeley, CA tJohns Hopkins University Asthma & Allergy Center, Baltimore, MD Recent studies have indicated that certain DNA sequences from bacteria contain CpG motifs which have immunostimulatory capabilities. Such immunostimulatory sequences (ISS) have been shown to