Abstract

We have described a group of cytokines that modulate mediator release from basophils and mast cells. Histamine releasing factors (HRF)-mediated activation of mast cells and basophils may represent a amplification mechanism of allergic process. The clinical importance of these cytokines can not be established until their chemical structure is better defined. We have purified one species of HRF (44 kD) by sequential HPLC. The objective of this grant is to purify this and other species of HRF to homogeneity and sequence them. In addition, we plan to raise monoclonal antibodies against HRF, develop immunoassays and affinity columns to aid purification. Crude supernatants containing HRF will be generated by culturing two cell lines, U937 and RPMI 8866, both of which produce HRF similar to that synthesized by mononuclear cells (MNC). Furthermore, MNC-derived HRF will be generated by culturing cells isolated from leukocytes concentrates obtained from Blood Bank donors. HRF will be purified by using sequential HPLC. We have recently demonstrated that a major fraction of HRF binds to heparin-agarose. We have also optimized the running conditions of a C4 reverse phase column, and recovered HRF from the column with satisfactory yield. The strategy for the purification of HRF is to apply sequential HPLC involving affinity, ion exchange and reverse phase columns. The homogeneity will be verified by one- and two-dimensional SDS-polyacrylamide gel electrophoresis. Purified HRF will be electroblotted onto Immobilon PVDF filters and used directly for sequencing. The latter will be accomplished using an updated Applied Bio-system Sequenator. Monoclonal antibodies will be raised in mice using purified HRF. Hybridoma cells will be grown in the peritoneum of pristane-treated mice. Purified antibody will be used to develop ELISA and/or RIA as well as affinity columns. Our final goal is to conduct a biochemical and immunological comparison of various species of HRF produced by different subpopulations of MNC. This will be accomplished by comparing the elution profile from HPLC columns and by testing the antigenic cross-reactivity among various species of HRF obtained from different sources.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI022940-06
Application #
3134671
Study Section
Immunological Sciences Study Section (IMS)
Project Start
1986-09-01
Project End
1994-03-31
Budget Start
1992-04-01
Budget End
1994-03-31
Support Year
6
Fiscal Year
1992
Total Cost
Indirect Cost

  Institution

Name
University of Texas Medical Br Galveston
Department
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555

  Publications

Sim, T C; Reece, L M; Hilsmeier, K A et al. (1995) Secretion of chemokines and other cytokines in allergen-induced nasal responses: inhibition by topical steroid treatment. Am J Respir Crit Care Med 152:927-33
Chonmaitree, T; Patel, J A; Lett-Brown, M A et al. (1994) Virus and bacteria enhance histamine production in middle ear fluids of children with acute otitis media. J Infect Dis 169:1265-70
Sim, T C; Grant, J A; Hilsmeier, K A et al. (1994) Proinflammatory cytokines in nasal secretions of allergic subjects after antigen challenge. Am J Respir Crit Care Med 149:339-44
Sim, T C; Hilsmeier, K A; Reece, L M et al. (1994) Interleukin-1 receptor antagonist protein inhibits the synthesis of IgE and proinflammatory cytokines by allergen-stimulated mononuclear cells. Am J Respir Cell Mol Biol 11:473-9
Alam, R; Dejarnatt, A; Stafford, S et al. (1993) Selective inhibition of the cutaneous late but not immediate allergic response to antigens by misoprostol, a PGE analog. Results of a double-blind, placebo-controlled randomized study. Am Rev Respir Dis 148:1066-70
Sim, T C; Alam, R; Forsythe, P A et al. (1992) Measurement of histamine-releasing factor activity in individual nasal washings: relationship with atopy, basophil response, and membrane-bound IgE. J Allergy Clin Immunol 89:1157-65
Alam, R; Lett-Brown, M A; Forsythe, P A et al. (1992) Monocyte chemotactic and activating factor is a potent histamine-releasing factor for basophils. J Clin Invest 89:723-8
Alam, R; Forsythe, P A; Stafford, S et al. (1992) Macrophage inflammatory protein-1 alpha activates basophils and mast cells. J Exp Med 176:781-6
Sim, T C; Hilsmeier, K A; Alam, R et al. (1992) Effect of topical corticosteroids on the recovery of histamine releasing factors in nasal washings of patients with allergic rhinitis. A double-blind, randomized, placebo-controlled study. Am Rev Respir Dis 145:1316-20
Alam, R; Forsythe, P A; Lett-Brown, M A et al. (1992) Interleukin-8 and RANTES inhibit basophil histamine release induced with monocyte chemotactic and activating factor/monocyte chemoattractant peptide-1 and histamine releasing factor. Am J Respir Cell Mol Biol 7:427-33

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