258 SFD-1alpha activates basophils via CYCR4

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Abstracts

258 SDF-lalpha

Activates Basophils Via CXCR4 Lors K. Poulsen. Tan Jinquan, Henrik H. Jacobi, Claw M. Reimert, Sha Quart. Steeu Dissing, PS Skov National University Hospital, Copenhagen, Denmark The CXC chemokine receptor 4 (CXCR4). a receptor for the chemokine stromal cell-derived factor-l alpha (SDF-la), has been found on T- and B-cells, dendritic and other cell types. but so far no reports have emerged on its expression on basophils. The aim of the present study was to elucidate whether CXCR4 is expressed on human basophils and if so whether its ligand. SDF-la, can activate basophils. Human basophils were purified from 50 ml EDTA-blood drawn from normal donors. A Percoll gradient step was followed by negative absorbtion by Dynabeads with specificity for CD3. CD4. CD8, CD14, CD15 and CD19. In general the yield was about I/? -1miocells of 85-95% purity. CXCR4 was identified by immunocytechemistry and flow cytometry. In a typical experiment using freshly isolated basophils 91.3% cells double-positive forCXCR4 and surface-bound IgE could be identified. In parallel, using real-time RT-PCR CXCR4 mRNA was found to be expressed in basophils purified to >99% by an additional positive selection on dynabeads coated with an antibody to the alpha-chain of the high affinity IgE receptor. By using a singlecell Calcium microscope it was demonstrated that 50 rig/ml but not 0.5 rig/ml of SDF- la induced an increase in intracellular free Ca2+ in basophils. Finally it was demonstrated that SDF-la activates basophils to chemotaxis and histamine release via CXCR4 on the cells, documented by tbe facts that anti-CXCRJ mAb but not murine IgG blocks SDF- la-induced chemotaxis and histamine release. Optimal concentrations of SDF-la were found to be around 100 rig/ml which resulted in a chemotactic index of 3 or more. For histamine release the optimal concentration of SDF-la was also 100 rig/ml which induced histamine release of 34-369. When the chemokines SDF- 1a, eotaxin. RANTES. MCP- I and MIP- I a were compared they demonstrated different potencies in induction of chemotaxis (eotaxin > SDF-la > RANTES >> MCP-1 >> MIP-la). and histamine release (MCP-1 >> SDF-la > eotaxin > RANTES > MIP-la). Interestingly. IL-3 priming has potentiated histamine release from basophils induced by these chemokines (except MIP-la). whereas, it did not change chemotactic migration pattern of basophils towards the chemokines. In conclusion, the SDF-la-CXCR4 pair seems to represent a new signal-activation system of the basophil. Interestingly, SDF-la has been described as a constitutive rather than an inflammatory chemokine. whereas we have here found that it induces chemotaxis and histamine release in basophils - processes that are usually associated with allergic reactions. This could indicate that the SDF-la CXCR4 pair is more important for inflammation than previously believed, and/or that the basophil may participate in other biological functions such as postinflammatory regenerative processes.

259

MIP3P, a B Cell Chemoattractant That Signals via a Pertussis Toxin Sensitive G Protein R Mil/ette*f#$i. Q Zhuang*f#$‘. B Mazer*fJ’ *Division of Allergy and Immunology tMontreal Children’s Hospital $Meakins Christie Laboratory §McGill University ‘Montreal, Canada BACKGROUND: Chemokines are potent agents that mediate chemoattraction and leukocyte homing during both homeostatic and inflammatory processes. A novel subset of chemokines is emerging characterized by constitutive expression within lymphoid tissues, unique chromosomal localization relative to other chemokines and

J ALLERGY CLIN IMMUNOL JANUARY 2000

specificity for lymphocytes. MIP-3P. a member of this group. is a B and T lymphocyte specific CC chemokine produced by activated antigen presenting cells in the paracortical, T cell rich zone of lymphoid tissues. It is the ligand of CCR7, a 7-membrane spanning G-protein coupled receptor. We have previously demonstrated that MIP-3P increases intracellular calcium concentrations in the LA350 B-lymphoblastoid cell line, suggesting that this cell line expresses CCR7 or a related protein. OBJECTIVE: The aim of the present study was to further elucidate the pathways involved in the cell-chemokine interaction and characterize the response of LA350 cells to differing concentrations of MIP-3P. METHODS: These studies employed the EBV transformed B cell line LA350. Intracellular Ca’+ concentrations were measured by spectrotlourimetry following cellular incubation with the fluorescent calcium chelator INDO-I-AM. Cellular chemotaxis was measured using the Boyden chamber. RESULTS: MIP-3P induced a rapid increase in intracellular calcium followed by a slow return towards baseline levels. Incubation with pertussis toxin inhibited this. confirming that the chemokine activates the cell in the characteristic manner of Ga,-protein-coupled receptors. Interestingly, the increases in intracellular calcium were demonstrated over a wide range of MIP-3P concentrations, from 5 rig/ml to 100 rig/ml. However, the elevation in intracellular Ca?+ was clearly dose dependent (47.7 ng/molM - 123.1 ng/molM). We have also assessed whether MIP3P could induce B cell chemotaxis. Our data suggests that indeed MIP-3P acts as a chemotactic molecule for LA350 cells and that its greatest effects occur at concentrations in the range of I rig/ml to 0. I nglml. CONCLUSION: It has been hypothesized that the production of MIP-3P by activated antigen presenting cells in the paracortical zone of the lymph nodes may retain the B cells within the T-rich zone favoring B-T cell interactions. This leads to B cell activation, proliferation and differentiation into memory or antibody secreting plasma cells. Our data suggests that MIP-3P activates B-cells. and induces chemoattraction at very low concentrations. This may be important in recruiting and retaining B-cells in the paracortical area. Future experiments using human peripheral B-cells in our system may help to further clarify the role of MIP-3P.

260 Identification

of a Novel Population of Mast Cells/Basophils in Human Periphal Blood of Allergic Patients Which Is HIV-I Susceptible Due to its Surface Expression of CD4, CCRS, and CXCR4 Y Li*. L Li*. R Waldq\‘*. S Reddel*. J Qi*. C Arch*. A Collins *, E Clark*, M Cooly\*, S Kouts*. N Hassanf, A Moharmnedi. A Cwtnir~ghaml’. G Wong#. R Stevens#. S Kri/is* *University of New South Wales and St George’s Hospital tuniversity of Sydney and Westmead Hospital *Harvard Medical School and Brigham and Women’s Hospital It is generally thought that human mast cells (MCs) and basophils are developmentally unrelated. Nevertheless. we recently identified an unusual population of metachromatic cells in the peripheral blood of three groups of patients with active allergic disorders. These cells resemble normal mature basophils in terms of their peripheral blood location, segmented nuclei, and their surface expression of the Bsp-I epitope but resemble normal mature MCs in terms of their surface expression of CD1 17 (c-kit) and their granule expression of the neutral proteases carboxypeptidase A, chymase. and tryptases alpha, I, Ii/beta, and/or III. We now report that these metachromatic cells store transmembrane tryptase in their granules. Moreover, they express on