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The human C3a receptor is expressed on neutrophils and monocytes, but not on B or T lymphocytes.

Martin U, Bock D, Arseniev L, Tornetta MA, Ames RS, Bautsch W, Köhl J, Ganser A, Klos A - J. Exp. Med. (1997)

Bottom Line: Flow cytometric analyses, functional assays, and binding assays on highly purified neutrophils confirmed C3aR expression and coupling.Monocytes were identified as an additional C3aR-positive cell population of the peripheral blood.In contrast, the receptor could not be detected on unchallenged B or T lymphocytes (or lymphocyte-derived Raji cells).

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Hannover Medical School, Hannover, Germany.

ABSTRACT
The pathophysiological relevance of the complement split product C3a as a proinflammatory mediator is still ill defined. The expression pattern of the human C3a receptor (C3aR) can provide important clues for the role of this anaphylatoxin in inflammation. There is strong evidence for C3aR expression on basophils, and eosinophils, but additionally, only on tumor cell lines of leukemic or hepatic origin. It is unclear whether neutrophils also express the C3aR, but need a costimulus provided by eosinophils for certain biological responses, or whether neutrophils lack the C3aR and respond to C3a via a secondary stimulus generated by eosinophils, i.e., by an indirect mode. In the present study, polyclonal antiserum raised against the second extracellular loop of the C3aR was used to characterize C3aR expression on peripheral blood leukocytes. For high degree purification of neutrophils, a negative selection method was established that decreased the contamination with CD9(bright+) eosinophils down to <0.2%. Flow cytometric analyses, functional assays, and binding assays on highly purified neutrophils confirmed C3aR expression and coupling. Monocytes were identified as an additional C3aR-positive cell population of the peripheral blood. The expression of the C3aR on eosinophils could be confirmed. In contrast, the receptor could not be detected on unchallenged B or T lymphocytes (or lymphocyte-derived Raji cells).

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Human monocytes respond inhomogeneously to C3a.  PBMCs (no granulocytes within 1,000 stained cells) were loaded with  Fluo3-AM as fluorescence indicator of free cytosolic Ca2+. Monocytes  were gated as indicated in the upper panels. The histograms of the fluorescence intensity (FL-1) resulting ∼10 s after stimulation with buffer or  20 nM of C5a, and increasing concentrations of C3a, respectively, are depicted. The response of monocytes to the C3a analogue peptide P117 was  similar (data not shown).
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Figure 5: Human monocytes respond inhomogeneously to C3a. PBMCs (no granulocytes within 1,000 stained cells) were loaded with Fluo3-AM as fluorescence indicator of free cytosolic Ca2+. Monocytes were gated as indicated in the upper panels. The histograms of the fluorescence intensity (FL-1) resulting ∼10 s after stimulation with buffer or 20 nM of C5a, and increasing concentrations of C3a, respectively, are depicted. The response of monocytes to the C3a analogue peptide P117 was similar (data not shown).

Mentions: The C3a-dependent Ca2+ increase in monocytes was determined flow cytometrically starting 10 s after application of the ligand, and compared with the response of monocytes to C5a. Diff-Quick™ staining revealed that no granulocytes were present within 1,000 cells of the PMNC preparation, and that all contaminating cells (∼80%) were of lymphocytic origin. For this functional assay, monocytes were gated within the PBMCs according to forward scatter and side scatter (Fig. 5, top). Flow cytometric analyses of an aliquot of this PMNCs preparation using CD14 (LPS receptor) as marker for monocytes confirmed that the selected gating was appropriate for monocytes (data not shown). The monocytes responded with an increase in Fluo3-determined fluorescence to a C3a stimulus, as compared with the buffer control (Fig. 5, bottom). As judged by the mean fluorescence values, the maximal C3a response of monocytes is reached at concentrations of C3a near 5–10 nM, similar to neutrophils (data not shown). Intriguingly, the monocytes did not respond homogeneously to C3a. With increasing C3a concentrations progressively more cells switched from a fluorescence value corresponding to the buffer control to a distinctly higher fluorescence state. However, even at maximal C3a concentrations a portion of the gated cell population did not show any increase in Ca2+. The same effect was observed after stimulation with the C3a analogue peptide P117. P252 served again as negative control (data not shown). In kinetic studies with increased incubation times after C3a application of up to 1 min (equivalent to the approximate duration of the response), a similar proportion of monocytes seemed to be non-C3a responsive (data not shown). In contrast, almost all gated cells responded homogeneously after stimulation using a maximal concentration of recombinant C5a (20 nM). These data would most likely suggest that there is a subpopulation of monocytes lacking the ability to react to C3a. These might correspond to the heterogeneous distribution of the C3aR as determined with the polyclonal anti-C3aR serum (see Fig. 2). If this is the case, it remains to be investigated whether the inhomogeneity actually reflects different subsets within monocytes or monocytes at different states of preactivation caused by the cell preparation. Similarly, functional subpopulations within neutrophils have been described for the IL-8 receptor (31). B or T lymphocytes did not react to C3a in this assay (data not shown). There is no evidence for C3aR expression on lymphocytes. Keeping in mind that no C3aR could be detected antigenetically, and that the response reaches its maximum already 10–15 s after stimulus application, it is very unlikely that lymphocytes stimulated by C3a would influence the Ca2+ response of monocytes.


The human C3a receptor is expressed on neutrophils and monocytes, but not on B or T lymphocytes.

Martin U, Bock D, Arseniev L, Tornetta MA, Ames RS, Bautsch W, Köhl J, Ganser A, Klos A - J. Exp. Med. (1997)

Human monocytes respond inhomogeneously to C3a.  PBMCs (no granulocytes within 1,000 stained cells) were loaded with  Fluo3-AM as fluorescence indicator of free cytosolic Ca2+. Monocytes  were gated as indicated in the upper panels. The histograms of the fluorescence intensity (FL-1) resulting ∼10 s after stimulation with buffer or  20 nM of C5a, and increasing concentrations of C3a, respectively, are depicted. The response of monocytes to the C3a analogue peptide P117 was  similar (data not shown).
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Figure 5: Human monocytes respond inhomogeneously to C3a. PBMCs (no granulocytes within 1,000 stained cells) were loaded with Fluo3-AM as fluorescence indicator of free cytosolic Ca2+. Monocytes were gated as indicated in the upper panels. The histograms of the fluorescence intensity (FL-1) resulting ∼10 s after stimulation with buffer or 20 nM of C5a, and increasing concentrations of C3a, respectively, are depicted. The response of monocytes to the C3a analogue peptide P117 was similar (data not shown).
Mentions: The C3a-dependent Ca2+ increase in monocytes was determined flow cytometrically starting 10 s after application of the ligand, and compared with the response of monocytes to C5a. Diff-Quick™ staining revealed that no granulocytes were present within 1,000 cells of the PMNC preparation, and that all contaminating cells (∼80%) were of lymphocytic origin. For this functional assay, monocytes were gated within the PBMCs according to forward scatter and side scatter (Fig. 5, top). Flow cytometric analyses of an aliquot of this PMNCs preparation using CD14 (LPS receptor) as marker for monocytes confirmed that the selected gating was appropriate for monocytes (data not shown). The monocytes responded with an increase in Fluo3-determined fluorescence to a C3a stimulus, as compared with the buffer control (Fig. 5, bottom). As judged by the mean fluorescence values, the maximal C3a response of monocytes is reached at concentrations of C3a near 5–10 nM, similar to neutrophils (data not shown). Intriguingly, the monocytes did not respond homogeneously to C3a. With increasing C3a concentrations progressively more cells switched from a fluorescence value corresponding to the buffer control to a distinctly higher fluorescence state. However, even at maximal C3a concentrations a portion of the gated cell population did not show any increase in Ca2+. The same effect was observed after stimulation with the C3a analogue peptide P117. P252 served again as negative control (data not shown). In kinetic studies with increased incubation times after C3a application of up to 1 min (equivalent to the approximate duration of the response), a similar proportion of monocytes seemed to be non-C3a responsive (data not shown). In contrast, almost all gated cells responded homogeneously after stimulation using a maximal concentration of recombinant C5a (20 nM). These data would most likely suggest that there is a subpopulation of monocytes lacking the ability to react to C3a. These might correspond to the heterogeneous distribution of the C3aR as determined with the polyclonal anti-C3aR serum (see Fig. 2). If this is the case, it remains to be investigated whether the inhomogeneity actually reflects different subsets within monocytes or monocytes at different states of preactivation caused by the cell preparation. Similarly, functional subpopulations within neutrophils have been described for the IL-8 receptor (31). B or T lymphocytes did not react to C3a in this assay (data not shown). There is no evidence for C3aR expression on lymphocytes. Keeping in mind that no C3aR could be detected antigenetically, and that the response reaches its maximum already 10–15 s after stimulus application, it is very unlikely that lymphocytes stimulated by C3a would influence the Ca2+ response of monocytes.

Bottom Line: Flow cytometric analyses, functional assays, and binding assays on highly purified neutrophils confirmed C3aR expression and coupling.Monocytes were identified as an additional C3aR-positive cell population of the peripheral blood.In contrast, the receptor could not be detected on unchallenged B or T lymphocytes (or lymphocyte-derived Raji cells).

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Hannover Medical School, Hannover, Germany.

ABSTRACT
The pathophysiological relevance of the complement split product C3a as a proinflammatory mediator is still ill defined. The expression pattern of the human C3a receptor (C3aR) can provide important clues for the role of this anaphylatoxin in inflammation. There is strong evidence for C3aR expression on basophils, and eosinophils, but additionally, only on tumor cell lines of leukemic or hepatic origin. It is unclear whether neutrophils also express the C3aR, but need a costimulus provided by eosinophils for certain biological responses, or whether neutrophils lack the C3aR and respond to C3a via a secondary stimulus generated by eosinophils, i.e., by an indirect mode. In the present study, polyclonal antiserum raised against the second extracellular loop of the C3aR was used to characterize C3aR expression on peripheral blood leukocytes. For high degree purification of neutrophils, a negative selection method was established that decreased the contamination with CD9(bright+) eosinophils down to <0.2%. Flow cytometric analyses, functional assays, and binding assays on highly purified neutrophils confirmed C3aR expression and coupling. Monocytes were identified as an additional C3aR-positive cell population of the peripheral blood. The expression of the C3aR on eosinophils could be confirmed. In contrast, the receptor could not be detected on unchallenged B or T lymphocytes (or lymphocyte-derived Raji cells).

Show MeSH
Related in: MedlinePlus