CD16b associates with high-density, detergent-resistant membranes in human neutrophils - PubMed (original) (raw)

CD16b associates with high-density, detergent-resistant membranes in human neutrophils

Maria J G Fernandes et al. Biochem J. 2006.

Abstract

CD16b is unique in that it is the only Fc receptor linked to the plasma membrane by a GPI (glycosylphosphatidylinositol) anchor. GPI-anchored proteins often preferentially localize to DRMs (detergent-resistant membranes) that are rich in sphingolipids and cholesterol and play an important role in signal transduction. Even though the responses to CD16b engagement have been intensively investigated, the importance of DRM integrity for CD16b signalling has not been characterized in human neutrophils. We provide direct evidence that CD16b constitutively partitions with both low- and high-density DRMs. Moreover, upon CD16b engagement, a significant increase in the amount of the receptor is observed in high-density DRMs. Similarly to CD16b, CD11b also resides in low- and high-density DRMs. In contrast with CD16b, the partitioning of CD11b in DRMs does not change in response to CD16b engagement. We also provide evidence for the implication of Syk in CD16b signalling and its partitioning to DRMs in resting and activated PMNs (polymorphonuclear neutrophils). Additionally, DRM-disrupting agents, such as nystatin and methyl-beta-cyclodextrin, alter cellular responses to CD16b receptor ligation. Notably, a significant increase in the mobilization of intracellular Ca2+ and in tyrosine phosphorylation of intracellular substrates after CD16b engagement is observed. Altogether, the results of this study provide evidence that high-density DRMs play a role in CD16b signalling in human neutrophils.

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Figures

Figure 1

Figure 1. Localization of CD16b to DRM-L in non-stimulated PMNs

PMNs were lysed in cold lysis buffer containing 1% Triton X-100 in the absence (−) or presence (+) of sodium carbonate and overlaid with a discontinuous sucrose gradient (as described in the Materials and methods section). Fractions were analysed by SDS/PAGE and immunoblotted with the anti-CD16 7.5.4 mAb. Fractions 1–4 contain soluble cellular components, fractions 11–15 contain DRM-L, and the remainder of the cellular components that partition in the pellet were loaded in lane P. Molecular mass sizes are given in kDa.

Figure 2

Figure 2. Localization of CD16b to DRM-L and DRM-H in non-stimulated PMNs

PMNs were lysed in cold lysis buffer containing 1% Triton X-100 and overlaid with a discontinuous gradient of OptiPrep (as described in the Materials and methods section). Fractions were analysed by SDS/PAGE and immunoblotted with an anti-flotillin-1 antibody (A) or the anti-CD16 7.5.4 mAb (B). Fractions 2–5 contain DRM-L, fractions 8 and 9 contain DRM-H, and fractions 10–15 contain the soluble cellular components. Molecular mass sizes are given in kDa.

Figure 3

Figure 3. Distribution of CD16b receptors in DRM-L and DRM-H in activated PMNs

(A) The kinetics of tyrosine phosphorylation after CD16b engagement were determined by incubating PMNs for 15 min at 4 °C with the 3G8 F(ab′)2 mAb followed by cross-linking with an anti-F(ab′)2 antibody for various periods of time at 22 °C. Samples were analysed by SDS/PAGE and immunoblotted with the 4G10 anti-phosphotyrosine mAb (αpY). (B) PMNs were activated for 1 min at 22 °C, as described in (A), lysed and overlaid with a sucrose gradient. (C) PMNs were incubated for 1 min at 37 °C with 3G8 F(ab′)2, cross-linked for 1 min at 37 °C, lysed in cold lysis buffer and overlaid with a discontinuous OptiPrep gradient. Fractions of non-activated (Resting) and activated (Activated) PMN were pooled (as described in the Results section), analysed by SDS/PAGE and immunoblotted with the anti-CD16 7.5.4 mAb. Molecular mass sizes are given in kDa. WB, Western blotting.

Figure 4

Figure 4. Distribution of CD16b at the plasma membrane of resting PMNs upon receptor engagement

Plasma membranes were isolated from PMNs and incubated without (Resting) or with (Activated) the 3G8 F(ab′)2 mAb and goat F(ab′2) anti-mouse F(ab′)2 as described in the Materials and methods section. Following the solubilization of the plasma membranes in 1% NP40 and centrifugation at 100000 g, the pellet (P) and supernatant (S) were analysed by SDS/PAGE and immunoblotted with the anti-CD16 7.5.4 mAb.

Figure 5

Figure 5. Distribution of CD11b and Syk in DRMs in resting and activated human PMNs

DRMs were isolated using an OptiPrep gradient from resting PMNs as described in Figure 2. Fractions were pooled (as described in the Results section) and analysed by SDS/PAGE and immunoblotted with the anti-CD11b (A) or anti-Syk (B) antibody. (C) The distribution of CD16b, Syk and CD11b in DRM-H was determined in non-activated (Resting) and activated (Activated) PMNs as described in Figure 3(C). The open bars represent DRM-H isolated from resting PMNs and the closed bars show DRM-H from activated PMNs. This Figure represents the results of six independent experiments. The statistical analysis was performed using Student's paired t test (_P_=0.041). NS, statistically non-significant; *, statistically significant.

Figure 6

Figure 6. Tyrosine-phosphorylation status of Syk upon CD16b cross-linking

Resting PMNs (−) or PMNs activated with the 3G8 F(ab′)2 mAb (+) were lysed and immunoprecipitated with an anti-Syk antibody under denaturing conditions (as described in the Materials and methods section). Immunoprecipitated proteins were analysed by SDS/PAGE and immunoblotted with the anti-phosphotyrosine 4G10 (αpY) or anti-Syk (αSyk) antibodies. IPP, immunoprecipitation; WB, Western blot.

Figure 7

Figure 7. Effects of nystatin and β-cyclodextrin on the tyrosine phosphorylation of intracellular proteins after CD16b engagement

(A) PMNs pre-incubated in HBSS containing 0.1% (v/v) DMSO or 30 μg/ml nystatin for 1 h at 37 °C were stimulated for various periods of time by cross-linking CD16b (1 min at 37 °C, as described in the Materials and methods section). The cross-linking reaction was stopped by lysing whole-cells directly in boiling 2×Laemmli's sample buffer. Samples were analysed by SDS/PAGE and immunoblotted with an anti-phosphotyrosine 4G10 mAb (αpY). (B) Similar experimental conditions as in (A) were used, with the exception that PMNs were incubated with 10 mM β-cyclodextrin at 37 °C for 30 min before CD16b cross-linking. Molecular mass sizes are given in kDa. WB, Western blot.

Figure 8

Figure 8. Effects of nystatin and β-cyclodextrin on the release of intracellular Ca2+ stores after CD16b engagement

(A) The release of Ca2+ stores was determined in PMNs stimulated with 3G8 mAb F(ab′)2 after a pre-incubation of 1 h in HBSS containing 0.1% (v/v) DMSO or 30 μg/ml nystatin (as described in the Materials and methods section). (B) Similar experimental conditions as in (A) were used, with the exception that PMNs were incubated with 10 mM β-cyclodextrin at 37 °C for 30 min before CD16b cross-linking.

Figure 9

Figure 9. Effects of methyl-β-cyclodextrin on the distribution of flotillin-1 and CD16b receptors in DRM-L and DRM-H in resting and activated PMNs

PMNs were incubated in HBSS (−) or in HBSS containing 10 mM methyl-β-cyclodextrin (+) at 37 °C for 30 min. CD16b was then cross-linked for 1 min at 37 °C (as described in the Materials and methods section.) with the 3G8 F(ab′)2 mAb (Activated). As a control, PMNs were also incubated in HBSS alone for 1 min at 37 °C (Resting). PMNs were then lysed in cold lysis buffer containing 1% Triton X-100 and overlaid with an OptiPrep gradient (as described in the Materials and methods section). Fractions were pooled (as described in the Results section) and analysed by SDS/PAGE and immunoblotted with an anti-flotillin-1 antibody (upper panel set) or with the anti-CD16b 7.5.4 mAb (lower panel set). MbCD, methyl-β-cyclodextrin. Molecular mass sizes are given in kDa.

References

    1. Gessner J. E., Heiken H., Tamm A., Schmidt R. E. The IgG Fc receptor family. Ann. Hematol. 1998;76:231–248. - PubMed
    1. Hulett M. D., Hogarth P. M. Molecular basis of Fc receptor function. Adv. Immunol. 1994;57:1–127. - PubMed
    1. Kimberly R. P., Salmon J. E., Edberg J. C. Receptors for immunoglobulin G. Molecular diversity and implications for disease. Arthritis Rheum. 1995;38:306–314. - PubMed
    1. Shen L., Guyre P. M., Fanger M. W. Polymorphonuclear leukocyte function triggered through the high affinity Fc receptor for monomeric IgG. J. Immunol. 1987;139:534–538. - PubMed
    1. Unkeless J. C., Shen Z., Lin C. W., DeBeus E. Function of human Fc γ RIIA and Fc γ RIIIB. Semin. Immunol. 1995;7:37–44. - PubMed

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