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Interaction between lymphocytes and inflammatory exudate cells. II. A proteolytic enzyme released by PMN as a
Abstract:
Mouse polymorphonuclear leukocytes (PMN) were harvested from the peritoneal cavity stimulated with sodium caseinate. The cells were cultivated in vitro and the supernatant of these cultures (SUP) was tested for enhancing potency on DNA synthesis by syngeneic thymocytes stimulated with phytohemagglutinin (PHA). The enhancing potency of the SUP was markedly influenced by duration of the donor cultures of PMN, population density of the cultures, and protein concentrations in the medium, respectively. The enhancing factor in the SUP was found to be non-dialysable, heat-labile, and stable in the pH ranging between 3 and 9 but labile in the pH below 2 or above 10; its m.w. was approximately 19.000 when measured by gel-filtration on Sephadex G-75. The factor had a proteolytic activity on 3H-acetyl hemoglobin (3HHb) at neutral pH (7.2). Both the proteolytic activity and thymocyte-helping potency of the SUP were similarly abolished by adding protease inhibitor (Trasylol) in soluble form, or by passing through a column of the inhibitor insolubilized. It was thus assumed that the enhancing effect of PMN on thymocyte response was associated with a neutral protease released from the cells: it may be termed a lymphocyte-helping protease (LHP).
Insights
Mouse polymorphonuclear leukocytes (PMN) release a factor that enhances thymocyte DNA synthesis. This lymphocyte-helping protease (LHP) is a neutral protease, crucial for immune cell communication.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Polymorphonuclear leukocytes (PMN) play a role in immune responses.
- Thymocytes are crucial for T-cell development and require specific signals for activation.
- Understanding intercellular communication is vital for immunology.
Purpose of the Study:
- To investigate the enhancing effect of PMN-derived factors on thymocyte DNA synthesis.
- To characterize the nature of the enhancing factor released by PMN.
- To determine if the enhancing factor possesses enzymatic activity.
Main Methods:
- Harvesting mouse peritoneal PMN stimulated with sodium caseinate.
- Culturing PMN in vitro and collecting the supernatant (SUP).
- Assessing SUP's effect on thymocyte DNA synthesis stimulated by phytohemagglutinin (PHA).
- Characterizing the factor's properties (dialysis, heat stability, pH stability, molecular weight via gel filtration).
- Testing for proteolytic activity using 3H-acetyl hemoglobin (3HHb).
- Evaluating the effect of protease inhibitors (Trasylol) on SUP activity.
Main Results:
- PMN supernatant (SUP) significantly enhanced thymocyte DNA synthesis.
- Enhancing potency was influenced by PMN culture duration, density, and protein concentration.
- The active factor was non-dialyzable, heat-labile, stable between pH 3-9, with a molecular weight around 19,000.
- The factor exhibited neutral proteolytic activity and this activity correlated with thymocyte-helping potency.
- Protease inhibitors abolished both proteolytic and enhancing activities.
Conclusions:
- A neutral protease, termed lymphocyte-helping protease (LHP), is released by PMN.
- LHP enhances thymocyte response, suggesting a role in immune cell communication.
- The findings link PMN proteolytic activity to their ability to modulate lymphocyte proliferation.