1. Cytokines in Asthma: Effects on Human Pulmonary Fibroblasts Shreya Lankala, Agostino Molteni, Betty Herndon UMKC School of Medicine Background & Rationale Asthma has long been considered a cellular immune problem Cytokines are small proteins made by cells in the immune system A chemokine is a type of cytokine that induces chemotaxis In the asthmatic patient, CD4+ T lymphocytes elevate chemokines in airway fluids and blood This study assayed effects of serum from asthmatic adults on the metabolism of healthy human lung fibroblasts in culture With sera from nonasthmatic volunteers (students/staff) as controls, the humoral effects on cultured human lung cells produced by asthmatic sera were measured Hypothesis: Cytokines present in severe asthma have toxic effects on pulmonary fibroblast mitochondria Methods Samples: Sera from asthmatic adults diagnosed by the Truman Medical Center Pulmonary Clinic (numbered, but without patient identification) and controls were sera from healthy nonasthmatic staff and medical students ELISA was performed to determine which serum samples contained the highest amount of IgE, a marker that is used to indicate the severity of asthma Results were analyzed by titer based on a 450 nm read of 2.000 or greater (high), a read of 0.25 (low) and a +/- read between those values WI38 human lung fibroblasts were obtained from ATCC and cultured in supplier recommended media (MEM) with 15% fetal calf serum For assay, cells were harvested, counted by hemocytometer (10 5 /mL) and added ~1,500 cells in 100 µL were added to wells of a 96 well plate, (usual media) Cells attached overnight, and media was replaced with MEM without serum In a sterile hood, asthma and control sera were added (15 µL) to each well according to a template, and the cells were covered and incubated at 37°C, 5% CO 2 for 24 hr. TACS© MTT Cell Proliferation Assay was performed by adding the MTT dye, a tetrazolium salt, 15 µL, to each well, then incubating overnight The tetrazolium salt starts as a golden yellow and stays yellow if cultured cells are not metabolizing well. Metabolizing cells (mitochondria) turn the media purple Intensity of the formazan (purple) shows cellular activity in the presence of the added sera, data were read at the purple wavelength Higher numbers suggest more viability (mitochondrial enzyme activity) ResultsSummary The design was to assay effects of serum from individuals with severe asthma on the metabolism of healthy human fibroblast cells in culture Chemokines, proteins that induce leukocyte activity in the asthmatic lung and which are present in asthmatic serum, play essential roles during immune and inflammatory responses It was our goal to determine if asthma has toxic effects to lung function due to these chemokines and if so, to what extent this damage occurs Sera from severe asthmatics and control sera from lung- healthy volunteers were tested on the same assay plate for comparison MTT assay of mitochondrial dehydrogenase activity of the cultured cells grown in presence of asthmatic or control sera showed significantly more response by fibroblasts to asthma Higher numbers suggest more viability so our study showed that asthmatic sera does not in fact cause increased toxicity to healthy pulmonary fibroblasts MTT of the asthmatics averaged 1.37 ± 0.12 vs 1.28 ± 0.11 for the controls, p=0.03 with 3 replications Since we did not have chart data on the asthmatic subject’s sera, high IgE and low IgE ELISA values were tested, summarized, and paired to MTT values for both groups IgE in the asthmatic population Human ELISA for IgE on our patient sera showed high IgE titers in wells 2, 7, 8, 10, 11, 12, 14, 16, 21, 22, 23, 24 MTT values for wells with high IgE titers was 1.378 ± 0.18 The MTT titer for ALL wells averaged was 1.376 Conclusion We concluded that there was no correlation between patient IgE and the MTT cell proliferation test. Cytokines present in asthma have not been shown to cause toxicity to pulmonary fibroblasts. Caveat to this finding: We have no information on how many of these “severe asthmatics” were taking steroid products which can significantly lower the IgE serum levels. Cytokines and chemokines have varied responses to steroid treatment. Importance to Medicine This study shows that expression of chemokines in serum of “severe asthmatic” patients was significantly greater than expression of chemokines in a lung-healthy population, p=0.03 The identity of the reactants in asthmatic sera is not yet known Other work, animal and human, have measured inflammatory markers in asthmatic fluids Other recent asthma research in humans 1-6 finds a delayed asthma response with significantly elevated serum chemokines A need exists to identify serum-borne reactants in asthmatic sera, and study on our model continues 1. Isgro M et al, Mucosal Immun. 2013, 6:718-27. 2. Sadik CD et al, J. Leukocyte Biol. 2012, 91:207-14. 3. Robroeks CMH et al, Clin. & Exp. Allergy 2010, 40:77-84. 4. Kawasaki, S. et al, J. Immunol. 2001, 166:2055-6,. 5. Afshar R. et al, J. Allergy & Clin. Immunol. 2013, 131:1644-52. 6. Xu S.Y. et al, Chin. Med. J. 2004, 117(1):30-6. References