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Granulocyte-macrophage colony-stimulating factor increases synthesis and expression of CR1 and CR3 by human
E Neuman1, J W Huleatt, R M Jack
1Department of Medicine, Harvard Medical School, Boston, MA.
Abstract:
Polymorphonuclear leukocytes (PMN) constitutively synthesize various plasma membrane proteins including CR1(3) (CD35), CR3 (or Mac-1) alpha-chain (CD11b) and MHC class I. PMN are also able to up-regulate rapidly the expression of CR1 and CR3 to the plasma membrane in response to agonists such as FMLP. To determine whether constitutive PMN translation was static or up-regulatable, PMN were cultured in the presence or absence of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) for 8 h. CR1, CR3 and class I proteins immunoprecipitated from lysates of 35S-methionine pulse-labeled PMN were resolved by SDS-PAGE, fluorographed and quantified by densitometry. GM-CSF-treated PMN synthesized 4.5-fold more class I protein, 3.7-fold more CR1, 2.4-fold more CD11b and 3.4-fold more CR3 beta-chain (CD18), compared with untreated control cells. Actinomycin D treatment of replicate samples of PMN decreased the amount of these proteins synthesized by each group of PMN from 30 to 90%, implying that continued translation was required for the increases in protein synthesis. Nascent CR and class I proteins were inserted into the plasma membrane of PMN, thereby supplementing the molecules already expressed on the cell surface. In addition to these longer term effects of GM-CSF, we observed its acute up-regulatory effects on PMN. GM-CSF induced a five- to 12-fold increase in the expression of CR1 and CR3 on the PMN cell surface within 30 min. These increases were both dose- and time-dependent with maximum up-regulation occurring at 25 pM and at 30 min. In contrast to the long term biosynthetic events, this rapid up-regulation was not dependent on protein synthesis but was due instead to mobilization of CR from intracellular compartments similar to those up-regulated by FMLP. These results demonstrate that PMN can respond to microenvironmental stimuli such as GM-CSF both by rapidly up-regulating and increasing translation and expression of functionally important plasma membrane proteins.
Insights
Polymorphonuclear leukocytes (PMN) increase synthesis of key membrane proteins when exposed to GM-CSF. This cytokine also rapidly mobilizes existing proteins to the cell surface, enhancing PMN function.
Area of Science:
- Immunology
- Cell Biology
- Hematology
Background:
- Polymorphonuclear leukocytes (PMN) constitutively express plasma membrane proteins like CR1, CR3, and MHC class I.
- PMN can rapidly up-regulate CR1 and CR3 expression upon stimulation by agonists such as FMLP.
Purpose of the Study:
- To investigate whether constitutive PMN translation is static or can be upregulated.
- To determine the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on PMN protein synthesis and surface expression.
Main Methods:
- PMN were cultured with or without GM-CSF for 8 hours.
- Proteins (CR1, CR3, MHC class I) were pulse-labeled with 35S-methionine, immunoprecipitated, resolved by SDS-PAGE, and quantified.
- Actinomycin D was used to assess the requirement for ongoing translation.
Main Results:
- GM-CSF treatment significantly increased the synthesis of class I, CR1, CD11b, and CR3 beta-chain (CD18) proteins.
- Increased protein synthesis was dependent on continued translation, as shown by Actinomycin D treatment.
- GM-CSF also induced rapid, dose- and time-dependent increases in CR1 and CR3 surface expression within 30 minutes, independent of protein synthesis, via mobilization from intracellular compartments.
Conclusions:
- PMN exhibit dual responses to GM-CSF: enhanced protein translation for long-term expression and rapid mobilization of existing proteins for acute responses.
- These findings highlight the plasticity of PMN in responding to microenvironmental cues to modulate immune functions.