Limits...
Transcellular delivery of vesicular SOCS proteins from macrophages to epithelial cells blunts inflammatory signaling.

Bourdonnay E, Zasłona Z, Penke LR, Speth JM, Schneider DJ, Przybranowski S, Swanson JA, Mancuso P, Freeman CM, Curtis JL, Peters-Golden M - J. Exp. Med. (2015)

Bottom Line: Secretion is tunable and occurs both in vitro and in vivo.SOCS secretion into lung lining fluid was diminished by cigarette smoking in humans and mice.Secretion and transcellular delivery of vesicular SOCS proteins thus represent a new model for the control of inflammatory signaling, which is subject to dysregulation during states of inflammation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine and Department of Microbiology and Immunology, University of Michigan Medical School; and Department of Environmental Health Sciences, School of Public Health; University of Michigan, Ann Arbor, MI 48109.

Show MeSH

Related in: MedlinePlus

SOCS3 secretion by AMs proceeds via an unconventional vesicular pathway and mainly involves MPs. (A) AMs were adhered and cultured for 1 h at 37°C or at 4°C. Then CM was concentrated and subjected to WB analysis for SOCS3. SOCS3 levels in CM are expressed as the percentage of SOCS3 secreted by AMs kept at 37°C. (B) AMs were treated with 1 µM monensin for 1 h, after which CM was harvested for determination of TNF by ELISA (left) or concentrated and subjected to WB analysis for SOCS3 (right). SOCS3 levels in CM are expressed as arbitrary densitometric units. (C) CM was obtained from AMs after 1-h adherence, concentrated, and incubated for 2 h with 0.1 mg/ml proteinase K in the presence or absence of 1% Triton X-100 and then analyzed by WB for SOCS3. SOCS3 is expressed as the percentage of that measured in nondetergent-treated CM. The dashed vertical line separates lanes that were loaded on the same gel but were not contiguous. (D) Neat CM and the flow through from a 0.2-µm filter were concentrated and subjected to either WB for SOCS3 or analysis by flow cytometry. Particles were further subjected to size determination using standard beads of known size. Additionally, MPs and Exos were purified from CM by differential centrifugation and subjected to WB for SOCS3. MPs were further analyzed for staining with FITC–annexin V and FITC–anti-SOCS3 with (continuous line) or without (dashed line) pretreatment with 0.2% NP-40. Additionally, whole CM, MPs, Exos, and vesicle-free CM (VFCM) were collected and then subjected to SOCS3 quantitation by ELISA (bottom graph). (E) AM plasma membranes were labeled by incubating cells on ice in the dark for 20 min with 100 µM of the fluorescent lipid 18:1-06:0 NBD PC (green) and counterstained with DAPI; then cells were washed twice with PBS and plated for 1 h, and MP budding was assessed by fluorescence microscopy using an Eclipse E600 microscope and 100 magnification; arrows indicate membrane blebs. (F) The MP pellet from AM CM was incubated with FITC–annexin V in the dark and imaged on a TE300 with a 60× oil immersion objective (NA 1.40, total magnification of 600). Data in A–D (except for ELISA data which are from a single experiment representative of two) represent the mean ± SE from at least three independent experiments; data in E and F are representative of two independent experiments. *, P < 0.05 versus 4°C cells (A), untreated cells (B), or CM untreated with 1% Triton X-100 (C).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4419346&req=5

fig2: SOCS3 secretion by AMs proceeds via an unconventional vesicular pathway and mainly involves MPs. (A) AMs were adhered and cultured for 1 h at 37°C or at 4°C. Then CM was concentrated and subjected to WB analysis for SOCS3. SOCS3 levels in CM are expressed as the percentage of SOCS3 secreted by AMs kept at 37°C. (B) AMs were treated with 1 µM monensin for 1 h, after which CM was harvested for determination of TNF by ELISA (left) or concentrated and subjected to WB analysis for SOCS3 (right). SOCS3 levels in CM are expressed as arbitrary densitometric units. (C) CM was obtained from AMs after 1-h adherence, concentrated, and incubated for 2 h with 0.1 mg/ml proteinase K in the presence or absence of 1% Triton X-100 and then analyzed by WB for SOCS3. SOCS3 is expressed as the percentage of that measured in nondetergent-treated CM. The dashed vertical line separates lanes that were loaded on the same gel but were not contiguous. (D) Neat CM and the flow through from a 0.2-µm filter were concentrated and subjected to either WB for SOCS3 or analysis by flow cytometry. Particles were further subjected to size determination using standard beads of known size. Additionally, MPs and Exos were purified from CM by differential centrifugation and subjected to WB for SOCS3. MPs were further analyzed for staining with FITC–annexin V and FITC–anti-SOCS3 with (continuous line) or without (dashed line) pretreatment with 0.2% NP-40. Additionally, whole CM, MPs, Exos, and vesicle-free CM (VFCM) were collected and then subjected to SOCS3 quantitation by ELISA (bottom graph). (E) AM plasma membranes were labeled by incubating cells on ice in the dark for 20 min with 100 µM of the fluorescent lipid 18:1-06:0 NBD PC (green) and counterstained with DAPI; then cells were washed twice with PBS and plated for 1 h, and MP budding was assessed by fluorescence microscopy using an Eclipse E600 microscope and 100 magnification; arrows indicate membrane blebs. (F) The MP pellet from AM CM was incubated with FITC–annexin V in the dark and imaged on a TE300 with a 60× oil immersion objective (NA 1.40, total magnification of 600). Data in A–D (except for ELISA data which are from a single experiment representative of two) represent the mean ± SE from at least three independent experiments; data in E and F are representative of two independent experiments. *, P < 0.05 versus 4°C cells (A), untreated cells (B), or CM untreated with 1% Triton X-100 (C).

Mentions: We found SOCS3 secretion to be unassociated with LDH release (not depicted), arguing against it being a manifestation of cytotoxicity. In addition, it was markedly reduced at 4°C, suggesting that it is an energy-dependent phenomenon (Fig. 2 A). To confirm that SOCS3 is indeed released by AMs through unconventional secretion, we tested the effects of monensin, an inhibitor of conventional secretion. As expected, monensin inhibited rat AM secretion of the known conventionally secreted protein TNF (Fig. 2 B, left); in contrast, it increased secretion of SOCS3 (Fig. 2 B, right), as it has previously been recognized to do for other unconventionally secreted proteins (Rubartelli et al., 1990). Similar results were obtained using brefeldin A, another inhibitor of conventional secretion (not depicted). Unconventional secretion can be vesicular in nature; the finding that SOCS3 in AM-derived CM was more sensitive to proteolysis in the presence of a detergent (Fig. 2 C) implied its packaging within a membranous structure, such as an extracellular vesicle.


Transcellular delivery of vesicular SOCS proteins from macrophages to epithelial cells blunts inflammatory signaling.

Bourdonnay E, Zasłona Z, Penke LR, Speth JM, Schneider DJ, Przybranowski S, Swanson JA, Mancuso P, Freeman CM, Curtis JL, Peters-Golden M - J. Exp. Med. (2015)

SOCS3 secretion by AMs proceeds via an unconventional vesicular pathway and mainly involves MPs. (A) AMs were adhered and cultured for 1 h at 37°C or at 4°C. Then CM was concentrated and subjected to WB analysis for SOCS3. SOCS3 levels in CM are expressed as the percentage of SOCS3 secreted by AMs kept at 37°C. (B) AMs were treated with 1 µM monensin for 1 h, after which CM was harvested for determination of TNF by ELISA (left) or concentrated and subjected to WB analysis for SOCS3 (right). SOCS3 levels in CM are expressed as arbitrary densitometric units. (C) CM was obtained from AMs after 1-h adherence, concentrated, and incubated for 2 h with 0.1 mg/ml proteinase K in the presence or absence of 1% Triton X-100 and then analyzed by WB for SOCS3. SOCS3 is expressed as the percentage of that measured in nondetergent-treated CM. The dashed vertical line separates lanes that were loaded on the same gel but were not contiguous. (D) Neat CM and the flow through from a 0.2-µm filter were concentrated and subjected to either WB for SOCS3 or analysis by flow cytometry. Particles were further subjected to size determination using standard beads of known size. Additionally, MPs and Exos were purified from CM by differential centrifugation and subjected to WB for SOCS3. MPs were further analyzed for staining with FITC–annexin V and FITC–anti-SOCS3 with (continuous line) or without (dashed line) pretreatment with 0.2% NP-40. Additionally, whole CM, MPs, Exos, and vesicle-free CM (VFCM) were collected and then subjected to SOCS3 quantitation by ELISA (bottom graph). (E) AM plasma membranes were labeled by incubating cells on ice in the dark for 20 min with 100 µM of the fluorescent lipid 18:1-06:0 NBD PC (green) and counterstained with DAPI; then cells were washed twice with PBS and plated for 1 h, and MP budding was assessed by fluorescence microscopy using an Eclipse E600 microscope and 100 magnification; arrows indicate membrane blebs. (F) The MP pellet from AM CM was incubated with FITC–annexin V in the dark and imaged on a TE300 with a 60× oil immersion objective (NA 1.40, total magnification of 600). Data in A–D (except for ELISA data which are from a single experiment representative of two) represent the mean ± SE from at least three independent experiments; data in E and F are representative of two independent experiments. *, P < 0.05 versus 4°C cells (A), untreated cells (B), or CM untreated with 1% Triton X-100 (C).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4419346&req=5

fig2: SOCS3 secretion by AMs proceeds via an unconventional vesicular pathway and mainly involves MPs. (A) AMs were adhered and cultured for 1 h at 37°C or at 4°C. Then CM was concentrated and subjected to WB analysis for SOCS3. SOCS3 levels in CM are expressed as the percentage of SOCS3 secreted by AMs kept at 37°C. (B) AMs were treated with 1 µM monensin for 1 h, after which CM was harvested for determination of TNF by ELISA (left) or concentrated and subjected to WB analysis for SOCS3 (right). SOCS3 levels in CM are expressed as arbitrary densitometric units. (C) CM was obtained from AMs after 1-h adherence, concentrated, and incubated for 2 h with 0.1 mg/ml proteinase K in the presence or absence of 1% Triton X-100 and then analyzed by WB for SOCS3. SOCS3 is expressed as the percentage of that measured in nondetergent-treated CM. The dashed vertical line separates lanes that were loaded on the same gel but were not contiguous. (D) Neat CM and the flow through from a 0.2-µm filter were concentrated and subjected to either WB for SOCS3 or analysis by flow cytometry. Particles were further subjected to size determination using standard beads of known size. Additionally, MPs and Exos were purified from CM by differential centrifugation and subjected to WB for SOCS3. MPs were further analyzed for staining with FITC–annexin V and FITC–anti-SOCS3 with (continuous line) or without (dashed line) pretreatment with 0.2% NP-40. Additionally, whole CM, MPs, Exos, and vesicle-free CM (VFCM) were collected and then subjected to SOCS3 quantitation by ELISA (bottom graph). (E) AM plasma membranes were labeled by incubating cells on ice in the dark for 20 min with 100 µM of the fluorescent lipid 18:1-06:0 NBD PC (green) and counterstained with DAPI; then cells were washed twice with PBS and plated for 1 h, and MP budding was assessed by fluorescence microscopy using an Eclipse E600 microscope and 100 magnification; arrows indicate membrane blebs. (F) The MP pellet from AM CM was incubated with FITC–annexin V in the dark and imaged on a TE300 with a 60× oil immersion objective (NA 1.40, total magnification of 600). Data in A–D (except for ELISA data which are from a single experiment representative of two) represent the mean ± SE from at least three independent experiments; data in E and F are representative of two independent experiments. *, P < 0.05 versus 4°C cells (A), untreated cells (B), or CM untreated with 1% Triton X-100 (C).
Mentions: We found SOCS3 secretion to be unassociated with LDH release (not depicted), arguing against it being a manifestation of cytotoxicity. In addition, it was markedly reduced at 4°C, suggesting that it is an energy-dependent phenomenon (Fig. 2 A). To confirm that SOCS3 is indeed released by AMs through unconventional secretion, we tested the effects of monensin, an inhibitor of conventional secretion. As expected, monensin inhibited rat AM secretion of the known conventionally secreted protein TNF (Fig. 2 B, left); in contrast, it increased secretion of SOCS3 (Fig. 2 B, right), as it has previously been recognized to do for other unconventionally secreted proteins (Rubartelli et al., 1990). Similar results were obtained using brefeldin A, another inhibitor of conventional secretion (not depicted). Unconventional secretion can be vesicular in nature; the finding that SOCS3 in AM-derived CM was more sensitive to proteolysis in the presence of a detergent (Fig. 2 C) implied its packaging within a membranous structure, such as an extracellular vesicle.

Bottom Line: Secretion is tunable and occurs both in vitro and in vivo.SOCS secretion into lung lining fluid was diminished by cigarette smoking in humans and mice.Secretion and transcellular delivery of vesicular SOCS proteins thus represent a new model for the control of inflammatory signaling, which is subject to dysregulation during states of inflammation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine and Department of Microbiology and Immunology, University of Michigan Medical School; and Department of Environmental Health Sciences, School of Public Health; University of Michigan, Ann Arbor, MI 48109.

Show MeSH
Related in: MedlinePlus