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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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C3aR mRNA is found in human monocytes but not in Raji  cells. Northern blot (a) and corresponding denaturing ethidium bromide– stained agarose gel (b) of poly(A+) RNA from nondifferentiated U937 cells  (Uφ) (∼6,500 C3aR per cell; reference 16), U937 cells with induction of  their C3aR expression by dibutyrylic cAMP (Ui) (∼30,000 per cell; references 15, 16), Raji cells (Ra), and monocytes (Mo), enriched from  PMNCs by adherence. The intensity of the ribosomal bands indicated  that similar concentrations of RNA were loaded in each lane. Additionally, the blot was rehybridized with a GAPDH probe as internal standard.
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Figure 1: C3aR mRNA is found in human monocytes but not in Raji cells. Northern blot (a) and corresponding denaturing ethidium bromide– stained agarose gel (b) of poly(A+) RNA from nondifferentiated U937 cells (Uφ) (∼6,500 C3aR per cell; reference 16), U937 cells with induction of their C3aR expression by dibutyrylic cAMP (Ui) (∼30,000 per cell; references 15, 16), Raji cells (Ra), and monocytes (Mo), enriched from PMNCs by adherence. The intensity of the ribosomal bands indicated that similar concentrations of RNA were loaded in each lane. Additionally, the blot was rehybridized with a GAPDH probe as internal standard.

Mentions: For flow cytometric analysis, a rabbit antiserum was raised against the second extracellular loop of the C3aR. Its optimal dilution of 1:4,000 was determined on a rat basophilic leukemia cell line (RBL-2H3 cells) stably expressing the human C3aR (7) and on nontransfected RBL cells (data not shown). Using these conditions, no difference between the fluorescence caused by immune, or preimmune sera, or the buffer control could be detected on Raji cells (data not shown). To confirm the thereby suggested absence of the C3aR on the cell surface of these cells, C3aR Northern blots with poly(A+) RNA from Raji cells were performed (Fig. 1). RNA from dibutyryl cAMP-induced (Boehringer, Mannheim, Germany) (Ui) and native U937 cells (Uφ) was applied as control. As described before (8), a prominent hybridization band at ∼2.4 kb, and a weak band at about 4 kb (16) could be detected. In contrast, even with 1 μg of poly(A+) RNA from Raji cells no C3aR transcript could be detected. The integrity of the Raji cell mRNA was affirmed by rehybridization with a probe for the housekeeping gene GAPDH (27), and the sharp rRNA bands in the corresponding gel. Consistent with these results, we could not detect any specific [125I]–hC3a binding on Raji cells (data not shown). The discrepancy to the results of Rogli ′ c et al. (9) who described C3aR expression on Raji cells antigenetically could be due to clonal differences of the analyzed cells, or the low serum dilution (1:200) used in their study. In our animals, such high concentrations result in high nonspecific binding of the preimmune serum on certain cell types.


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)

C3aR mRNA is found in human monocytes but not in Raji  cells. Northern blot (a) and corresponding denaturing ethidium bromide– stained agarose gel (b) of poly(A+) RNA from nondifferentiated U937 cells  (Uφ) (∼6,500 C3aR per cell; reference 16), U937 cells with induction of  their C3aR expression by dibutyrylic cAMP (Ui) (∼30,000 per cell; references 15, 16), Raji cells (Ra), and monocytes (Mo), enriched from  PMNCs by adherence. The intensity of the ribosomal bands indicated  that similar concentrations of RNA were loaded in each lane. Additionally, the blot was rehybridized with a GAPDH probe as internal standard.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198980&req=5

Figure 1: C3aR mRNA is found in human monocytes but not in Raji cells. Northern blot (a) and corresponding denaturing ethidium bromide– stained agarose gel (b) of poly(A+) RNA from nondifferentiated U937 cells (Uφ) (∼6,500 C3aR per cell; reference 16), U937 cells with induction of their C3aR expression by dibutyrylic cAMP (Ui) (∼30,000 per cell; references 15, 16), Raji cells (Ra), and monocytes (Mo), enriched from PMNCs by adherence. The intensity of the ribosomal bands indicated that similar concentrations of RNA were loaded in each lane. Additionally, the blot was rehybridized with a GAPDH probe as internal standard.
Mentions: For flow cytometric analysis, a rabbit antiserum was raised against the second extracellular loop of the C3aR. Its optimal dilution of 1:4,000 was determined on a rat basophilic leukemia cell line (RBL-2H3 cells) stably expressing the human C3aR (7) and on nontransfected RBL cells (data not shown). Using these conditions, no difference between the fluorescence caused by immune, or preimmune sera, or the buffer control could be detected on Raji cells (data not shown). To confirm the thereby suggested absence of the C3aR on the cell surface of these cells, C3aR Northern blots with poly(A+) RNA from Raji cells were performed (Fig. 1). RNA from dibutyryl cAMP-induced (Boehringer, Mannheim, Germany) (Ui) and native U937 cells (Uφ) was applied as control. As described before (8), a prominent hybridization band at ∼2.4 kb, and a weak band at about 4 kb (16) could be detected. In contrast, even with 1 μg of poly(A+) RNA from Raji cells no C3aR transcript could be detected. The integrity of the Raji cell mRNA was affirmed by rehybridization with a probe for the housekeeping gene GAPDH (27), and the sharp rRNA bands in the corresponding gel. Consistent with these results, we could not detect any specific [125I]–hC3a binding on Raji cells (data not shown). The discrepancy to the results of Rogli ′ c et al. (9) who described C3aR expression on Raji cells antigenetically could be due to clonal differences of the analyzed cells, or the low serum dilution (1:200) used in their study. In our animals, such high concentrations result in high nonspecific binding of the preimmune serum on certain cell types.

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