J ALLERGY CLIN IMMUNOL

FEBRUARY 2010

AB178 Abstracts

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696 Activation of Mast Cells and their Subsets in the Synovium inOsteoarthritis (OA) and Rheumatoid Arthritis (RA)

X. Zhou1, N. S. Abdullah1, R. Gobezie2, D. M. Lee3, A. F. Walls1; 1Uni-

versity of Southampton, Southampton, UNITED KINGDOM, 2Case

Western Reserve University, Cleveland, OH, 3Brigham & Women’s Hos-

pital, Boston, MA.

RATIONALE: Mast cells are abundant cellular constituents of the syno-

vium. Alterations in relative numbers of mast cell subpopulations (defined

according to the presence of absence of chymase or carboxypeptidase)

have been noted in the arthritic synovium, but the extent to which mast

cell subsets may be activated is not known.

METHODS: Synovial fluid (SF) was collected from patients with OA

(n594), early OA (29) and RA (31) and from healthy volunteers (16).

ELISA for mast cell tryptase, carboxypeptidase and chymase were devel-

oped and validated for use with SF. Levels of matrix metalloprotease

(MMP) 2 and 9 were assessed by gelatine zymography.

RESULTS: Levels of carboxypeptidase and chymase detected in SF from

OA and RA cases were substantially higher than in healthy subjects (p <

0.001), but not those with early OA. Tryptase was detected in all SF sam-

ples, but concentrations were not elevated in the arthritic groups compared

with healthy controls; and tryptase levels were not associated with carbox-

ypeptidase or chymase concentrations. On the other hand, levels of carbox-

ypeptidase were strongly correlated with those of chymase in each disease

group. Carboxypeptidase and chymase concentrations were both associ-

ated with MMP2 and MMP9 levels in SF, and in the RA group with blood

levels of rheumatoid factor and the erythrocyte sedimentation rate.

CONCLUSIONS: Mast cell activation is a feature of both OA and RA.

Carboxypeptidase, chymase and tryptase show distinct patterns of release

and clearance in SF, and deserve attention as biomarkers in arthritis.

697 Alpha Tryptase: Potential Roles in Inflammation Distinct fromthose of b-tryptase

A. M. Abdelmotelb1, M. E. Khedr1, S. L. F. Pender1, X. Zhou1, C. P. Som-

merhoff2, J. W. Holloway1, A. F. Walls1; 1University of Southampton,

Southampton, UNITED KINGDOM, 2Ludwig Maximilians University,

Munich, GERMANY.

RATIONALE: Tryptases are among the most abundant products of the hu-

man mast cell. Beta-tryptase has emerged as an important mediator of al-

lergic inflammation. The copy number of a-tryptase has recently been

found to be associated with the asthma phenotype, but the function of

this allelic variant to b-tryptase is unknown. We have investigated potential

actions of a-tryptase.

METHODS: C57BL/6 mice were injected intra-peritoneally with recom-

binant a or b-tryptases (0.005 or 0.5 ug/mouse; 12 mice per group). After

6, 12 or 24 h, mice were killed and peritoneal lavage performed.

Inflammatory cells were enumerated and levels of albumin and total pro-

tein determined. Gelatine zymography was applied to examine the activity

of matrix metalloprotease (MMP)-2 and MMP-9. In separate experiments,

cells of the human bronchial epithelial line 16HBE were incubated with

tryptases and expression of mRNA for IL-8, IL-6 and TNF-a examined

by quantitative PCR.

RESULTS: Injection of a-tryptase induced the accumulation of neutro-

phils, eosinophils, macrophages and mast cells (p<0.01) but not lympho-

cytes. Under the same conditions, b-tryptase stimulated greater

neutrophil recruitment, but the accumulation of other cell types was less

marked. MMP-9 activity in lavage fluid was unaffected in a-tryptase in-

jected mice, but it was increased in those injected with b-tryptase.

Following addition of a-tryptase to epithelial cells, there was down-regu-

lation of mRNA for IL-6 and TNF-a (p<0.05) whereas b-tryptase induced

strong up-regulation of the cytokines investigated.

CONCLUSION: Recombinant a-tryptase may be a stimulus for the re-

cruitment of inflammatory cells and altered cytokine gene expression

with effects different from those of b-tryptase.

698 Characterization of Putative Basophil Nanotobues byConfocal Microscopy

A. Praslick, M. Masilimani, M. Gross, W. G. Shreffler; Mount Sinai

School of Medicine, New York, NY.

RATIONALE: Basophils have been implicated in a wider variety of

immune functions than previously recognized, including direct or indirect

antigen presentation. We sought to better characterize our previous observa-

tion that activation induced the formation of filamentous cell structures.

METHODS: Human basophils were enriched from buffy coats using mag-

netic beads and activated in a polarized fashion with anti-FcER1 conju-

gated beads or uncoated control beads on poly-L-lysine coated

coverslips. Parallel cultures were fixed every 5 minutes and cells were

stained with DAPI and phalloidin. Images were acquired on a Leica

TCS-SP5. Quantitative data were obtained using Leica software.

RESULTS: Stimulated basophils were observed to frequently produce ac-

tin-positive filamentous structures, approximately 80 nm at their smallest

diameter, and often containing dilated sections, bifurcations and pseudo-

pod-like tips. The structures could be fully formed by 5 minutes. Two dis-

tinct activation phenotypes were quantified: cells with nanotubes >2 cell

diameters in length, or cells displaying cytoskeletal rearrangement without

nanotube formation. At 20 minutes, 28% of basophils stimulated with anti-

FcERI beads formed nanotubes and 28% appeared activated without nano-

tube formation (n5371). In contrast, 6% of control stimulated basophils

formed nanotubes, and 12% displayed the activated phenotype without

nanotubes (n5387). The putative nanotubes averaged 31 mm in length

(3X average cell diameter) in anti-FcERI treated cells, and 21 mm with

control beads.

CONCLUSIONS: To our knowledge the formation of these structures by

basophils has not been reported. As nanotubes in other leukocytes have

been demonstrated to function in intercellular communication, their ex-

pression in basophils may play a role in antigen presentation.

699 Degranulation, Cytokine mRNA Expression and SV40Transfection of an SCF-independent Human Mast Cell Line(USF-MC1)

M. C. Glaum1, G. Liu1, J. Wang1, R. F. Lockey2, S. S. Mohapatra2; 1Uni-

versity of South Florida, Tampa, FL, 2James A. Haley Veteran’s Hospital,

Tampa, FL.

RATIONALE: A human cord blood (HUCB)-derived, stem cell factor

(SCF)-independent human mast cell line (USF-MC1) has been previously

described. Cytokine expression from USF-MC1 is now compared to the

LAD2 mast cell line. In addition, immortalization of USF-MC1 with

pCMV-SV40-LT is explored.

METHODS: Stem cells were isolated from HUCB using CD133 bead

selection, and cultured as previously described. Cells were primed with bi-

otinylated human myeloma IgE (100 ng/ml) and challenged with buffer

(control), streptavidin (100 ng/ml), ionomycin (2.5 mM) or atrial natriuretic

peptide (ANP) (100 ng/ml). TcRNAwas examined for expression of IL-12

receptor (R) b, IL-10, TNFSF4, IL-17, TSLP and TNF-a by real-time PCR.

USF-MC1 cells were transfected with pCMV-SV40-LT and expression of

LT was confirmed by RT-PCR and western blot. Expression of CD117

and FceR1 was detected by flow cytometry. The presence of tryptase and

chymase was evaluated by immunofluorescent staining, and degranulation

was measured by beta-hexosaminidase (b-Hex) release.

RESULTS: In USF-MC1 and LAD2 cells, IgE-crosslinking, ionomycin

and ANP-challenge induced maximal mRNA expression of IL-12Rb,

IL10, TNFSF4, IL-17, TSLP and TNF-a at 6hr. Patterns of mRNA expres-

sion in USF-MC1 paralleled the cytokine expression patterns observed in

LAD2. Stable transfection of USF-MC1 with pCMV-SV40-LT was ob-

served with intact expression of FceR1, CD117, tryptase, chymase, and

similar amounts of b-Hex release were measured in transfected vs. non-

transfected USF-MC1 cells.

CONCLUSIONS: USF-MC1 expresses select cytokine mRNAs in re-

sponse to IgE-mediated and IgE-independent stimuli in a comparable

way to LAD2. Transfection of USF-MC1 with pCMV-SV40-LT may pro-

vide a unique model for study of human mast cell activation.