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Azurophilic granules of human neutrophils contain CD14
D A Rodeberg1, R E Morris, G F Babcock
1Shriners Burns Institute, Department of Cell Biology, University of Cincinnati College of Medicine, Ohio 45229-3095, USA.
Abstract:
CD14, the leukocyte receptor for lipopolysaccharide (LPS), is important in the response of human polymorphonuclear leukocytes (PMNs) to infection with gram-negative bacteria. The level of CD14 on the PMN surface increases after exposure to some inflammatory stimuli such as N-formyl-methionyl-leucyl-phenylalanine (fMLP). These newly expressed CD14 molecules probably come from an intracellular pool of preformed receptors. We sought to further characterize PMN CD14 expression, upregulation, and shedding and to define the intracellular location of CD14 molecules. Our results demonstrate that both LPS and fMLP significantly increased CD14 cell surface expression; however, neither phorbol myristate acetate (PMA) or A23187 increased receptor levels on the PMN surface. Neither fMLP, PMA, or A23187 stimulated the release of soluble CD14 from PMNs. Intracellular CD14 was observed in >90% of PMNs examined by flow cytometry and confocal microscopy. Additional analyses using CD14 enzyme-linked immunosorbent assays and electron microscopy studies, examining PMN granules separated by discontinuous sucrose or Percoll gradients, showed that CD14 was present in both the plasma membrane-secretory vesicle fractions and azurophilic granules.
Insights
Lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) increase CD14 expression on human polymorphonuclear leukocytes (PMNs). Intracellular CD14 is found in PMNs, particularly within azurophilic granules and the plasma membrane.
Area of Science:
- Immunology
- Cell Biology
Background:
- CD14 is a leukocyte receptor crucial for recognizing lipopolysaccharide (LPS) from gram-negative bacteria.
- CD14 expression on polymorphonuclear leukocytes (PMNs) can be upregulated by inflammatory stimuli like N-formyl-methionyl-leucyl-phenylalanine (fMLP).
- Upregulated CD14 is thought to originate from an intracellular reservoir within PMNs.
Purpose of the Study:
- To investigate the expression, upregulation, and shedding of CD14 on PMNs.
- To determine the intracellular localization of CD14 molecules within PMNs.
Main Methods:
- Flow cytometry and confocal microscopy were used to assess CD14 expression and localization.
- Enzyme-linked immunosorbent assays (ELISAs) quantified CD14 levels.
- Electron microscopy and density gradient centrifugation analyzed CD14 presence in PMN granules and cellular fractions.
Main Results:
- Both LPS and fMLP significantly increased CD14 surface expression on PMNs.
- Phorbol myristate acetate (PMA) and A23187 did not increase PMN surface CD14 levels.
- Neither fMLP, PMA, nor A23187 induced the release of soluble CD14.
- Intracellular CD14 was detected in over 90% of PMNs.
- CD14 was localized to the plasma membrane, secretory vesicles, and azurophilic granules.
Conclusions:
- LPS and fMLP are key stimuli for CD14 upregulation on PMN surfaces.
- PMNs possess a significant intracellular pool of CD14, primarily located in azurophilic granules and associated with the plasma membrane.
- CD14 shedding is not significantly induced by fMLP, PMA, or A23187 in PMNs.