Transcript Research Template - UMKC School of Medicine

Cytokines in Asthma: Effects on Human Pulmonary Fibroblasts
Shreya Lankala, Agostino Molteni, Betty Herndon
UMKC School of Medicine
Background & Rationale
Results
Summary
•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 (105
/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% CO2 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)
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.
•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 lunghealthy 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
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
References
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.