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HRF of 30 kDa: evidence for active synthesis
1Centre de Recherche en Inflammation et Immunologie-Rhumatologie, Le Centre Hospitalier de l'Université Laval, Sainte-Foy, Québec, Canada.
Abstract:
A variety of mediators are involved in the pathophysiology of a number of clinical conditions. Among them histamine-releasing factors (HRF) act as secretagogue for basophils and mast cells to cause cell degranulation and histamine release. Studies on the kinetics of HRF production by activated mononuclear cells (MNC) have suggested an active synthesis of this cytokine. Using a [35S]-metabolic cell labeling method, we first analyzed the capacity of MNC from patients with allergic rhinitis to ragweed to actively synthesize HRF. We then found that stimulation of MNC with allergen promoted a synthesis of large quantities of proteins, including a protein of about 30 kDa. Secondly, we developed a method for the enrichment of HRF molecules from crude supernatants of ragweed-stimulated MNC using ion exchange and gel filtration chromatography. We observed that the yield of HRF activity is seen just before the chymotrypsinogen marker. The estimated molecular weight was 30-35 kDa.
Insights
Histamine-releasing factors (HRF) are actively synthesized by mononuclear cells (MNC) from allergic rhinitis patients upon allergen stimulation. This study characterized HRF production and purification, revealing its role in allergic responses.
Area of Science:
- Immunology
- Molecular Biology
- Allergy Research
Background:
- Histamine-releasing factors (HRF) are key mediators in allergic conditions, triggering histamine release from mast cells and basophils.
- Previous studies suggested that activated mononuclear cells (MNC) actively synthesize HRF, but detailed characterization was lacking.
Purpose of the Study:
- To investigate the active synthesis of HRF by MNC from patients with allergic rhinitis.
- To develop a method for enriching and characterizing HRF molecules.
Main Methods:
- Utilized [35S]-metabolic cell labeling to assess de novo protein synthesis in MNC stimulated with ragweed allergen.
- Employed ion exchange and gel filtration chromatography for HRF enrichment from cell supernatants.
- Determined HRF molecular weight and activity yield.
Main Results:
- MNC from allergic rhinitis patients actively synthesized significant amounts of protein, including a ~30 kDa protein, upon allergen stimulation.
- HRF activity was successfully enriched and found to elute near the chymotrypsinogen marker.
- The estimated molecular weight of the purified HRF was 30-35 kDa.
Conclusions:
- Mononuclear cells actively synthesize histamine-releasing factors in response to allergens.
- This research provides a method for HRF enrichment and characterization, aiding further study into allergic pathophysiology.