Related Experiment Videos
Substance P production by P388D1 macrophages: a possible autocrine function for this neuropeptide
1Department of Physiology and Biophysics, University of Alabama, Birmingham.
Abstract:
This study investigates the ability of P388D1 macrophages to synthesize and secrete substance P (SP). Using a monoclonal anti-SP antibody (termed MASP-1) coupled to Sepharose, it was possible to immunoaffinity purify from culture supernates a peptide that was antigenically related to SP. P388D1 macrophage cultured in the presence of 35S-methionine secreted into culture supernates a labelled peptide which could be recognized by MASP-1. Affinity-purified, P388D1-derived SP was shown to be chemically similar to synthetic SP using gel-filtration chromatography and reverse-phase HPLC. In addition, an RIA using a polyclonal, monospecific antibody was used to quantify the amount of secreted SP in cultured supernates. P388D1 macrophages secreted 222 pg SP per 10(8) cells, whereas SP secretion by control thymocyte cultures was not detectable. The functionality of the P388D1-derived SP was also investigated. Since exogenously added SP can increase secretion of an interleukin-1 (IL-1)-like activity in these cells, we questioned whether an anti-SP antibody could remove P388D1-secreted SP from the culture, and in turn reduce cytokine production. By culturing varying dilutions of MASP-1 with P388D1 cells, it was possible to decrease cytokine production by P388D1 cells compared to cultures containing no antibody or with normal mouse immunoglobulin G (IgG). Taken together, these studies demonstrate that macrophage-derived SP may function in an autocrine or paracrine fashion to modulate macrophage function.
Insights
P388D1 macrophages synthesize and secrete substance P (SP), a neuropeptide. This secreted SP influences cytokine production, suggesting a role in modulating macrophage function.
Area of Science:
- Immunology
- Neuroscience
- Cell Biology
Background:
- Macrophages play a crucial role in immune responses.
- Substance P (SP) is a neuropeptide involved in various physiological processes.
- The role of SP in macrophage function is not fully understood.
Purpose of the Study:
- To investigate the synthesis and secretion of SP by P388D1 macrophages.
- To determine the functional significance of macrophage-derived SP in cytokine production.
Main Methods:
- Immunoaffinity purification using anti-SP antibody (MASP-1).
- Radiolabeling with 35S-methionine to track peptide secretion.
- Gel-filtration chromatography and reverse-phase HPLC for chemical characterization.
- Radioimmunoassay (RIA) for quantifying SP secretion.
- Functional assays involving anti-SP antibody to assess cytokine modulation.
Main Results:
- P388D1 macrophages synthesize and secrete SP.
- Secreted SP is chemically similar to synthetic SP.
- P388D1 macrophages secrete 222 pg SP per 10(8) cells.
- Anti-SP antibody treatment reduced cytokine production by P388D1 cells.
Conclusions:
- Macrophage-derived SP can be synthesized and secreted.
- Secreted SP modulates macrophage function, potentially via autocrine or paracrine mechanisms.
- This finding highlights a novel role for SP in immune cell communication.