Cytokines in Asthma: Effects on Human Pulmonary Fibroblasts Shreya Lankala, Agostino Molteni, Betty...
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Cytokines in Asthma: Effects on Human Pulmonary Fibroblasts Shreya Lankala, Agostino Molteni, Betty Herndon UMKC School of Medicine Background & Rationale
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 L 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 L) to each
well according to a template, and the cells were covered and
incubated at 37C, 5% CO 2 for 24 hr. TACS MTT Cell Proliferation
Assay was performed by adding the MTT dye, a tetrazolium salt, 15
L, 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 subjects 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