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Feb 2020

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In vitro Measurement of Membrane Attack Complex in RPE Cells
视网膜色素上皮细胞膜攻击复合物的体外测定   

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Abstract

Initiation of the complement system results in the formation of a multiprotein pore termed the membrane attack complex (MAC, C5b-C9). MAC pores accumulate on a cell surface and can result in cell lysis. The retinal pigment epithelium (RPE) is a single monolayer of pigmented epithelial cells located at the posterior poll of the eye that forms the outer blood retinal barrier. RPE cells are highly polarized with apical microvilli and basolateral contact with Bruch’s membrane. In order to obtain biologically relevant polarized RPE cultures in vitro, RPE cells are seeded onto the apical side of a transwell filter and cultured for 4 weeks in low serum media. MAC formation on RPE cells has been reported to be sub-lytic. MAC formation can be achieved in vitro by introduction of normal human serum (NHS) to media following serum starvation for 24 h. NHS contains all serum complement proteins required to initiate complement activation and MAC formation. We combined in vitro RPE polarization and complement activation to visualize MAC formation in vitro utilizing confocal microscopy allowing for high resolution MAC imaging.

Keywords: Membrane attack complex (MAC) (攻膜复合体), Retinal pigment epithelium (RPE) (视网膜色素上皮), Normal human serum (NHS) (正常人血清)

Background

The Complement system is an evolutionary conserved innate immune pathway. There are three major independent yet overlapping pathways for complement activation that converge at the C3 convertase, the classical, the lectin and the alternative pathways. In the classical pathway, immune complexes (Antigen-Antibody complexes) bind C1 via the C1q subcomponent, then the C1s protease subunit cleaves complement factors C4 and C2. Fragments of these (C4bC2b) forms an enzyme complex ‘C3 convertase’, that cleaves C3 to C3b and releases the anaphylatoxin, C3a. The binding of C3b to the C3 convertase generates the C5 convertase (C4bC2bC3b). The lectin pathway is an analogous system, except that the initiating step is the binding by lectins to repetitive sugars on microbial surfaces. Mannose-associated serine proteases (MASPs) take the place of the C1 proteases. The alternative pathway (AP) continuously self-activates at a low level to generate C3b that deposits on pathogens or debris. C3b or C3(H2O) engages the rate limiting alternative pathway components, factors B (FB) and D (FD), to form the alternative C3 convertase (C3bBb), which in turn cleaves more C3 into C3a and C3b. The binding by another C3b to the C3 convertase generates the C5 convertase (C3bBbC3b). Properdin (P) is a positive regulator that stabilizes both the AP C3 and C5 convertases. The C5c convertase subsequently cleaves C5 to release the potent anaphylatoxin C5a, while C5b engages the terminal pathway and initiates the formation of the membrane attack complex (MAC). In this way activation of the complement system results in the assembly of transmembrane pores; MAC (Figure 1). MAC formation on gram-negative bacteria causes cell lysis. In nucleated cells MAC deposition can result in cell death by apoptosis (Nauta et al., 2002), direct lysis (Koski et al., 1983) or in some cases MAC can be sub-lytic and promote an inflammatory response (Niculescu and Rus, 2001). The number of MAC pores which form on a cells surface is directly related to cell lysis and as such MAC formation is tightly regulated by complement inhibitors. CD59 is a direct inhibitor of complement and is expressed on the cell surface of various host tissues (Meri et al., 1991). In addition to expression of complement inhibitors some nucleated cells can endocytose or exocytose MAC pores to prevent cell lysis (Morgan et al., 1987).



Figure 1. The three independent complement activating pathways leading to C3 convertase activation


Aberrant activation of complement and increased MAC formation is involved in the pathogenesis of age-related macular degeneration. MAC accumulation has been observed in donor eye tissue and is most abundant in the choroid in choriocapillaris endothelial cells (Mullins et al., 2014). MAC deposition is also observed although less readily on the retinal pigment epithelium (RPE). This may be explained by the finding that the RPE can rapidly endocytose and clear MAC pores (Georgiannakis et al., 2015). Sub-lytic MAC does not appear to be responsible for RPE cell death, however, it may act as a pro-inflammatory signaling mechanism contributing to overactivation of the innate immune system and leading ultimately to tissue degeneration (Mulfaul et al., 2020).


The RPE is a highly specialized monolayer of pigmented epithelial cells located between the neural retina and the choriocapillaris. In vivo, the RPE forms polarized monolayers with apical microvilli which interconnect with the overlying photoreceptor outer segments. The basolateral RPE has basal infoldings in close contact with Bruch’s membrane. The RPE is therefore an essential component of the outer blood retinal barrier and is necessary for maintenance of healthy vision. Monolayer polarization is essential to RPE function. In order to achieve a polarized monolayer of RPE cells in vitro, cells can be seeded on transwell filters and cultured for at least 4 weeks (Figure 2). This results in the formation of apical microvilli and polarized expression of tight junction proteins (Kannan et al., 2006; Sonoda et al., 2009). Polarizing RPE in vitro allows us to closer mimic RPE behavior in the mammalian eye.


In order to study the contribution of MAC formation on the RPE in AMD we first polarized both the immortalized RPE cell line ARPE-19 or primary human fetal RPE (hfRPE). We then incubated cells with normal human serum (NHS) which contains all of the necessary components to drive complement activation and MAC deposition. NHS alone resulted in no visible MAC formation on ARPE-19 cells and only a small number of MAC were observed on hfRPE cells (Mulfaul et al., 2020). The apparent lack of MAC on RPE cells in spite of the presence of NHS is likely due to RPE expression of complement regulators including CD59 which directly inhibits MAC complex formation (Yang et al., 2009). Furthermore, if MAC complex’s do form the RPE can efficiently remove MAC by internalization of the MAC pore by endocytosis (Georgiannakis et al., 2015). We found that stimulation of RPE with an inflammatory insult prior to supplementation with NHS allowed MAC pores to persist on RPE cells that could be imaged by confocal microscopy. In a previous report, we demonstrated that the oxidative protein modification 2-(ω-carboxyethyl)pyrrole CEP, which is found in abundance in AMD donors (Gu et al., 2003) and is known to activate TLR2 and CD36, increased complement activation and MAC deposition on the RPE (Mulfaul et al., 2020). RPE cells activated with CEP in the presence of complement rich serum accumulate immunofluorescent measurable MAC pores. This protocol can be used as an in vitro model to study whether therapeutic targets can reduce MAC deposition on the RPE as we have previously demonstrated using an anti-TLR2 inhibitor (Mulfaul et al., 2020).

Materials and Reagents

  1. T75 cell culture flasks (Corning, catalog number: CLS3275)

  2. 15 ml conical tube (Corning, catalog number: CLS430791)

  3. 0.4 μm polyester transwell inserts (VWR, catalog number: 734-1581)

  4. Polylysine coated slide (Thermo Fisher Scientific, catalog number: 10219280).

  5. ARPE-19 cells (ATCC, catalog number: CRL-2302)

  6. Alexa Fluor 488 Goat Anti-Rabbit (Thermo Fisher Scientific, catalog number: A11034)

  7. Normal Goat Serum (Sigma-Aldrich, catalog number: G9023-10ml)

  8. Collagen IV (Sigma-Aldrich, catalog number: C5533)

  9. DMEM/F-12 Ham (Sigma-Aldrich, catalog number: D8437-500ML)

  10. Fetal Bovine Serum (Sigma-Aldrich, catalog number: F9665-500ML)

  11. Pennicillin Streptomycin (Sigma-Aldrich, catalog number: P4333-100ML)

  12. Phosphate Buffered Saline (Sigma-Aldrich, catalog number: D8662-500ML)

  13. Trypsin-EDTA (Biosciences, catalog number: 25200-056)

  14. Normal human serum (Sigma-Aldrich, catalog number: H4522-20ml)

  15. ZO-1 (Thermo Fisher Scientific, catalog number: 40-2200)

  16. Anti-C5b-9 (Santa Cruz Biotech, clone ae11, catalog number: sc58935)

  17. Hoechst (Sigma-Aldrich, catalog number: B2261-25MG)

  18. Mowiol® 4-88 (Sigma-Aldrich, catalog number: 81381-50G)

  19. Glycerol (Sigma-Aldrich, catalog number: G5516)

  20. Tris-HCl (Thermo Fisher Scientific, catalog number: 10724344)

  21. Paraformaldehyde (Sigma-Aldrich, catalog number: 158127)

  22. NaOH (Sigma-Aldrich, catalog number 567530)

  23. Triton X-100 (Sigma-Aldrich, catalog number: x100-500ML)

  24. L-glutamine (Thermo Fisher Scientific, catalog number: 10378-016)

  25. Mowiol 4-88 (see Recipes)

  26. 4% PFA (see Recipes)

Equipment

  1. Fume hood

  2. Hemocytometer

  3. -80 °C freezer

  4. 4 °C fridge

  5. Tweezers

  6. Biological Safety Cabinet Class ll

  7. Heat Block

  8. Confocal laser scanning microscope Axio Observer Z1 Inverted Microscope equipped with a Zeiss LSM 700 T-PMT Scanning unit and a 40x plan.

Procedure

  1. Transwell preparation

    1. Coat 0.4 μm polyester transwell inserts with 100 μg/ml of collagen IV in PBS by pipetting the appropriate volume of collagen onto the apical side of the transwell filter.

    2. Leave the transwell filters to air dry with the lid off in a cell culture grade biosafety cabinet.

    3. Gently aspirate off any additional collagen from each transwell filter.

    4. Wash each filter twice in PBS prior to cell seeding.


  1. Cell seeding ARPE-19

    1. ARPE-19 cells are maintained in DMEM/F-12 Ham with 10% FBS and 1% PS in T75 cell culture flasks. Cells are passaged at 80% confluency (we have used them up to passage 30).

    2. Aspirate medium from T75 flask and wash cell monolayer twice with 5 ml of sterile PBS.

    3. Add 3 ml of pre-warmed trypsin and incubate at 37 °C for 5 min until cells begin to detach.

    4. Add 3 ml of DMEM F-12.

    5. Use a 10 ml stripette to aspirate 6 ml of trypsin/media and flush the bottom wall of the T75 to remove all attached cells.

    6. Transfer cell suspension to a 15 ml conical tube.

    7. Pellet cells by centrifugation at 205 × g for 5 min.

    8. Gently aspirate media.

    9. Resuspend cell pellet in 1 ml of fresh media.

    10. Count cells using a hemocytometer.

    11. Pipette 600 μl of DMEM/F-12 Ham containing 10% FBS into the basolateral side of the transwell filter.

    12. Seed cells at a density of 1.7 × 105 cells per cm2 in the upper transwell chamber in 200 μl of DMEM/F-12 Ham containing 10% FBS, (transwell surface area 0.33 cm2 per well).

    13. Two days later replace media with DMEM/F-12 containing 1% FBS.

    14. Replace media every Monday, Wednesday and Friday for 4 weeks (Figure 2).



      Figure 2. Polarization of RPE cells grown on transwell filters. A. Schematic representation of the RPE. Polarised RPE cells display apical microvilli and ZO-1 tight junction protein expression. B. RPE cells seeded on the apical side of a transmembrane filter and maintained for at least 4 weeks form polarised monolayers as indicated by defined ZO-1 immunohistochemistry in both (C) ARPE-19 cells and (D) primary hfRPE cells. Scale bars = 20 µm.


  2. Cell seeding primary human fetal RPE (hfRPE) cells

    1. Cells were kindly provided by Dr. Arvydas Maminishkis from the National Eye Institute (NEI), Bethesda, USA, and were received as a confluent monolayer of P-0 (Maminishkis et al., 2006).

    2. hfRPE cells are received in a T25 with filled with media.

    3. After receiving gently aspirate all media from the cell monolayer.

    4. Add 5 ml of fresh media Alpha MEM (w/o L-glutamine Lonza BE12-169F), plus Pen/strep + L-glutamine, 5% FBS.

    5. Leave cells to rest for at least 1 day prior to plating.

    6. Aspirate cell culture media.

    7. Wash cells with 50 ml of PBS.

      Note: This will fill the entire flask.

    8. Remove 50 ml of PBS and repeat for a second wash step.

    9. Pipette 5 ml of warm trypsin-EDTA into the flask.

    10. Place flask in the incubator for 15 min.

    11. Use a spinal needle with a 3 ml syringe to aspirate the trypsin from the RPE monolayer.

    12. Direct the flow of the needle against the wall of the flask which has adherent cells.

    13. Press down on the syringe to stream wash the cells from the wall of the flask.

    14. Use a 5 ml pipette to collect the cell suspension into a sterile conical tube.

    15. Immediately add RPE media containing 15% FBS to the cells in the conical tube.

    16. Repeat steps 9-15 a second time to ensure all cells are dislodged.

    17. Pellet cells by centrifugation at 205 × g for 5 min.

    18. Gently aspirate media.

    19. Resuspend cell pellet in 1 ml of fresh media (no need for FCS).

    20. Count cells using a hemocytometer.

    21. Add 600 μl of RPE media containing 15% FCS to the basolateral well of each transwell.

    22. Seed cells at a density of 100 × 103 cells per cm2 into the apical transwell chamber in 200 μl of RPE media containing 15% FCS, transwell surface area 0.33 cm2 per well.

    23. 48 h later aspirate media from both apical and basolateral transwell chambers.

    24. Pipette 200 μl of RPE media containing 5% FCS into the apical and 600 μl into the basolateral chamber.

    25. Continue to change RPE media every Monday, Wednesday and Friday for 4 weeks.


  3. Validation of tight junction formation

    Note: Validation of tight junction formation can be conducted in parallel with the MAC formation assay.

    1. After 4 weeks in culture RPE cells on transwell inserts are fixed by adding 200 μl of 4% paraformaldehyde (PFA) to the apical membrane and 600 μl of 4% PFA to the basolateral chamber.

    2. Cells are incubated in PFA for 10 min at room temperate.

    3. Gently aspirate PFA taking care not to poke a hole in the transwell filter or disturb the cells.

    4. Wash cells on filter 3 times for 5 min with 200 μl of PBS in the apical compartment and 600 μl on the basolateral side.

    5. Pipette 200 μl or 600 μl of 0.05% Triton X-100 into the apical and basolateral chambers respectively.

    6. Cells are incubated in 0.05% Triton X-100 for 10 min at room temperature to permeabilize the cell membrane.

    7. Aspirate Triton X-100.

    8. Pipette 200 μl or 600 μl of 5% normal goat serum (NGS) into the apical and basolateral chambers respectively.

    9. Block cells in NGS for 1 h at room temperature.

    10. Aspirate 5% NGS.

    11. Pipette 100 μl of ZO-1 antibody (1:100 in 5% NGS) into the apical side of the transwell filter.

    12. 100 μl of antibody was pipetted into the bottom of an empty 6-well plate and the transwell inserts were placed to sit directly on the antibody.

    13. Inserts were incubated overnight at 4 °C.

    14. Aspirate antibody from apical chamber.

    15. Place transwell into a new plate with 600 μl of PBS in the basolateral chamber and pipette 100 μl of PBS into the apical side.

    16. Repeat this wash step 3 times.

    17. Pipette 100 μl or 600 μl of PBS with goat anti-rabbit 488 (1:500) into the apical and basolateral chambers respectively.

    18. Incubate transwells in secondary antibody for 2 h at room temperature.

    19. Aspirate off secondary antibody.

    20. Pipette 200 μl or 600 μl of PBS into the apical and basolateral chambers respectively.

    21. Repeat PBS wash 3 times.

    22. Aspirate off last PBS

    23. Add 200 μl of Hoechst (1:10,000) to the apical transwell.

    24. Incubate transwell for 2 min.

    25. Wash transwells 3 times in PBS.

    26. Gently remove all liquid from the transwell filter.

    27. Turn the transwell upside down on a clean surface.

    28. Gently use a sterile scalpel to cut around the circumference of the membrane.

    29. Use a sterile tweezer to grip one side of the filter after the first incision.

    30. Gently use the tweezers to loosen the membrane as you continue to cut slowly with the blade. Take care not to scratch the cell monolayer with the tweezers.

    31. Once the membrane is cut free use the tweezers to mount the membrane basolateral side down on to a polylysine coated slide. i.e., cells should be facing up on top of the slide.

    32. Pipette 50-100 μl of Mowiol® 4-88 mounting media onto the membrane and cover with cover slip.


  4. MAC formation Assay

    1. 48 h prior to MAC formation assay remove all RPE media from both apical and basolateral transwell chambers.

    2. Pipette 600 μl of serum free DMEM/F-12 Ham into the basolateral transwell chamber.

    3. Pipette 200 μl of serum free DMEM/F-12 Ham into the apical chamber.

    4. Thaw stock bottle of normal human serum (NHS) rapidly at 37 °C.

    5. Aliquot NHS into single use aliquots (300-500 μl).

    6. Store aliquoted NHS at -80 °C.

    7. On the day of the MAC assay remove two aliquots of NHS from -80 °C.

    8. Thaw aliquots on ice.

    9. Heat inactivate one aliquot of NHS by placing in either a heat block or water bath set to 56 °C.

    10. Incubate serum for 30 min at 56 °C inverting the tube every 10 min.

    11. Allow the heat inactivated serum to cool to room temperature.

    12. Designate which transwells will be used for apical or basolateral MAC formation.

    13. Aspirate media from transwells.

    14. Add 600 μl of serum free media to the basolateral chamber and 180 μl of serum free media to the apical chamber of wells designated for apical MAC assessment.

    15. Add 540 μl of serum free media to the basolateral chamber and 200 μl of serum free media to the apical chamber of wells designated for basolateral MAC assessment.

    16. Pipette 20 μl of either NHS or heat-inactivated (Hi) HiNHS to the apical chamber of wells containing 180 μl of media.

    17. Pipette 60 μl of either NHS or HiNHS to the basolateral chamber of wells containing 540 μl of media.

    18. Incubate RPE with NHS or HiNHS for 2-24 h.

    19. After 24 h fix cells by adding 200 μl of 4% PFA to the apical membrane and 600 μl of 4% PFA basolateral chamber.

    20. Incubate cells in PFA for 10 min at room temperature.

    21. Gently aspirate PFA taking care not to poke a hole in the transwell filter or disturb the cells.

    22. Wash cells on filter 3 times for 5 min with 200 μl of PBS in the apical compartment and 600 μl on the basolateral side.

    23. Pipette 200 μl or 600 μl of 5% bovine serum albumin (BSA) into the apical and basolateral chambers respectively.

    24. Block cells in BSA for 1 h at room temperature.

    25. Aspirate 5% BSA.

    26. Pipette 100 μl of C5b-9 MAC antibody (1:25 in 5% BSA) into the apical side of the transwell filter.

    27. 100 μl of antibody was pipetted into the bottom of an empty 6-well plate and the transwell inserts were placed to sit directly on the antibody, the plate is lidded and sealed with parafilm.

    28. Inserts were incubated overnight at 4 °C in a fridge.

    29. Aspirate antibody from apical chamber.

    30. Place transwell into a new plate with 600 μl of PBS in the basolateral chamber and pipette 100 μl of PBS into the apical side.

    31. Repeat this wash step 3 times.

    32. Pipette 100 μl or 600 μl of PBS with goat anti-mouse 647 (1:500) and phalloidin (1:500) into the apical and basolateral chambers respectively.

    33. Incubate transwells in secondary antibody for 2 h at room temperature.

    34. Aspirate off secondary antibody.

    35. Pipette 200 μl or 600 μl of PBS into the apical and basolateral chambers respectively.

    36. Repeat PBS wash 3 times.

    37. Aspirate off last PBS

    38. Add 200 μl of Hoechst (1:10,000) to the apical transwell.

    39. Incubate transwell for 2 min.

    40. Wash transwells 3 times in PBS.

    41. Gently remove all liquid from the transwell filter.

    42. Turn the transwell upside down on a clean surface.

    43. Gently use a sterile scalpel to cut around the circumference of the membrane.

    44. Use a sterile tweezer to grip one side of the filter after the first incision.

    45. Gently use the tweezers to loosen the membrane as you continue to cut slowly with the blade. Take care not to scratch the cell monolayer with the tweezers.

    46. Once the membrane is cut free use the tweezers to mount the membrane on to a polylysine coated slide (Thermo Scientific).

    47. Pipette 50-100 μl of Mowiol® 4-88 mounting media onto the membrane and cover with cover slip.

    48. MAC formation on ARPE-19 cells (Figure 3) and hfRPE cells (Figure 4) was imaged using a confocal laser scanning microscope.



      Figure 3. MAC assay on ARPE-19 cells. MAC formation (red) was observed in ARPE-19 cells stimulated with CEP (that induces the alternative complement pathway) and 10% NHS as a source of complement for 24 h. No staining was observed in the NHS alone or HiNHS control. Scale bar = 20 μm.



      Figure 4. MAC Assay on hfRPE. MAC formation (green) was observed in hfRPE cells stimulated with CEP and 10% NHS as a source of complement for 2 h. No staining was observed in the NHS alone or HiNHS controls. Scale bar = 20 μm.

Data analysis

Staining was analysed using a confocal laser scanning microscope Axio Observer Z1 inverted microscope equipped with a Zeiss LSM 700 T-PMT scanning unit and a 40× plan.

Recipes

  1. Mowiol 4-88

    2.4 g Mowiol

    6 g glycerol

    6 ml H2O

    Stir for several hours at room temperature

    Add 12 ml 0.2 M Tris-HCl (pH 8.5) and heat to 50 °C for 10 min

    Centrifuge Mowiol at 5,000 × g for 15 min

    Aliquot and store at -20 °C

  2. 4% PFA

    1. Add 4 g PFA to 50 ml of H2O

    2. Add 1 ml of 1 M NaOH, stir gently on a heating block at ~60 °C until PFA is dissolved

    3. Add 10 ml of 10× PBS and allow the mixture to cool to room temperature

    4. Adjust pH to 7.4

    5. Adjust the final volume to 100 ml with H2O

    6. Filter the solution through a 0.45-μm membrane filter to remove any particulate matter

    7. Make the PFA solution fresh prior to use, or store in aliquots at −20 °C for several months

    Notes:

    1. Avoid repeated freeze/thawing.

    2. Preparation of PFA can give off toxic fumes, use a fume hood as a precautionary measure.

Acknowledgments

This research was funded by the BrightFocus Foundation (M2016030), United States, Health Research Board Ireland (HRB-HRA/2013.290) and Science Foundation Ireland (SFI 15/CDA/3497), Royal Victoria Eye and Ear Hospital (RVEEH), Ireland, National Children's Research Centre (NCRC) , Ireland, Irish Research Council (IRCLA/2017/295), Ireland. The original research paper that displayed this protocol was published in cell reports (Mulfaul et al., 2020).

Competing interests

Authors have no financial or non-financial competing interests.

References

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  2. Gu, X., Meer, S. G., Miyagi, M., Rayborn, M. E., Hollyfield, J. G., Crabb, J. W. and Salomon, R. G. (2003). Carboxyethylpyrrole protein adducts and autoantibodies, biomarkers for age-related macular degeneration. J Biol Chem 278(43): 42027-42035.
  3. Kannan, R., Zhang, N., Sreekumar, P. G., Spee, C. K., Rodriguez, A., Barron, E. and Hinton, D. R. (2006). Stimulation of apical and basolateral VEGF-A and VEGF-C secretion by oxidative stress in polarized retinal pigment epithelial cells. Mol Vis 12: 1649-1659.
  4. Koski, C. L., Ramm, L. E., Hammer, C. H., Mayer, M. M. and Shin, M. L. (1983). Cytolysis of nucleated cells by complement: cell death displays multi-hit characteristics. Proc Natl Acad Sci U S A 80(12): 3816-3820.
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  13. Yang, P., Tyrrell, J., Han, I. and Jaffe, G. J. (2009). Expression and modulation of RPE cell membrane complement regulatory proteins. Invest Ophthalmol Vis Sci 50(7): 3473-3481.

简介

[摘要]补体系统的启动导致形成称为膜攻击复合物(MAC,C5b-C9)的多蛋白孔。MAC孔积聚在细胞表面,可导致细胞裂解。视网膜色素上皮细胞(RPE)是位于眼那种形式的后轮询色素上皮细胞的单个单层š外血视网膜屏障。RPE细胞高度极化,顶端微绒毛和与Bruch膜的基底外侧接触。为了在体外获得生物学上相关的极化RPE培养物,将RPE细胞接种到Transwell滤膜的顶端,并在低血清培养基中培养4周。MAC形成Ò Ñ据报道,RPE细胞是亚裂解的。通过在血清饥饿24小时后向培养基中引入正常人血清(NHS),可以在体外实现MAC的形成。NHS包含启动补体激活和MAC形成所需的所有血清补体蛋白。我们结合了体外RPE极化和补体激活,以利用共聚焦显微镜在体外可视化MAC形成,从而实现了高分辨率MAC成像。


[背景]补体系统是一种进化保守的先天免疫途径。补体激活存在三种主要的独立但重叠的途径,它们在C3转化酶,经典途径,凝集素途径和替代途径中收敛。在经典途径中,免疫复合物(抗原-抗体复合物)通过C1q亚成分结合C1,然后C1s蛋白酶亚基裂解补体因子C4和C2。这些片段(C4bC2b)形成酶复合物“ C3转化酶”,将C3裂解为C3b并释放出过敏毒素C3a。C3b与C3转化酶的结合产生C5转化酶(C4bC2bC3b)。凝集素途径是类似的系统,除了起始步骤是凝集素与微生物表面上的重复糖结合。甘露糖相关的丝氨酸蛋白酶(MASP)代替了C1蛋白酶。替代途径(AP)在低水平连续自我激活,以生成C3b,该C3b沉积在病原体或碎片上。C3b或C3(H 2 O)与限速替代途径组分B(FB)和D(FD)结合,形成替代C3转化酶(C3bBb),后者依次将更多的C3裂解为C3a和C3b。另一C3b与C3转化酶的结合产生C5转化酶(C3bBbC3b)。备解素(P)是可稳定AP C3和C5转化酶的正调节剂。C5c转化酶随后切割C5以释放有效的过敏毒素C5a,而C5b参与末端途径并启动膜攻击复合物(MAC)的形成。这样,补体系统的活化导致跨膜孔的组装。MAC (图1)。革兰氏阴性细菌上的M AC形成会引起细胞裂解。在有核细胞的MAC沉积可导致细胞死亡的细胞凋亡(诺塔等人,2002) ,直接裂解(考斯基等人,1983)或在某些情况下可将MAC子-裂解和促进炎性反应(尼古列斯库和罗斯,2001)。在细胞表面上形成的MAC孔的数量与细胞裂解直接相关,因此MAC的形成受到补体抑制剂的严格调控。CD59是补体的直接抑制剂,并在各种宿主组织的细胞表面表达(Meri等,1991)。除了表达补体抑制剂外,一些有核细胞还可以内吞或胞吐出MAC孔以防止细胞溶解(Morgan等,1987)。


图1 。导致C3转化酶激活的三个独立的补体激活途径


和补体的异常活化增加MAC的形成参与的发病一个GE -相关米acular变性。MAC积累已在供体眼组织中观察到,并且在脉络膜毛细血管内皮细胞的脉络膜中含量最高(Mullins等,2014)。尽管不易在视网膜色素上皮(RPE)上观察到MAC沉积。这可以通过以下发现来解释:RPE可以快速内吞并清除MAC孔(Georgiannakis等,2015)。亚裂解的MAC似乎不是RPE细胞死亡的原因,但是,它可能是促炎性信号传导机制,有助于先天免疫系统的过度激活并最终导致组织变性(Mulfaul等,2020)。

RPE是位于神经视网膜和脉络膜毛细血管之间的高度专门化的色素上皮细胞单层。在体内,RPE与顶部微绒毛形成极化的d单层,这些微绒毛与上面的感光器外部片段相互连接。基底外侧RPE具有与布鲁赫膜紧密接触的基底皱褶。因此,RPE是视网膜血外屏障的重要组成部分,对于维持健康的视力是必不可少的。莫nolayer极化是RPE功能是必不可少的。为了在体外获得极化的RPE细胞单层,可将细胞接种在Transwell滤膜上并培养至少4周(图2)。这导致顶端微绒毛的形成和紧密连接蛋白的极化表达(Kannan等,2006;Sonoda等,2009)。偏光RPE体外允许小号我们能够在哺乳动物的眼睛更接近模仿RPE行为。

为了研究MAC形成对AMD中RPE的贡献,我们首先极化了永生化的RPE细胞系ARPE-19或原代人胎儿RPE(hfRPE )。然后,我们将细胞与正常人血清(NHS)一起孵育,其中含有驱动补体激活和MAC沉积的所有必需成分。单独的NHS导致在ARPE-19细胞上没有可见的MAC形成,并且在hfRPE细胞上仅观察到少量的MAC (Mulfaul等人,2020)。尽管存在NHS,但RPE细胞上MAC仍明显缺乏,这可能是由于RPE表达补体调节剂(包括直接抑制MAC复合物形成的CD59)所致(Yang等,2009)。此外,如果形成MAC复合物,则RPE可以通过内吞作用通过MAC孔的内在化而有效地去除MAC (Georgiannakis等人,2015)。我们发现,在补充NHS之前用炎性刺激刺激RPE可使MAC孔在共聚焦显微镜可以成像的RPE细胞上持续存在。在以前的报告中,我们证实了氧化蛋白修饰的2-(ω-羧乙基)吡咯CEP在AMD供体中大量存在(Gu等,2003),并且已知它可以激活TLR2和CD36,从而增加补体激活。和在RPE上的MAC沉积(Mulfaul等人,2020年)。在富含补体的血清存在下,被CEP激活的RPE细胞积聚了免疫荧光可测量的MAC孔。如我们先前使用抗TLR2抑制剂所证实的,该方案可以用作体外模型以研究治疗靶标是否可以减少MAC在RPE上的沉积(Mulfaul等人,2020)。

关键字:攻膜复合体, 视网膜色素上皮, 正常人血清

材料和试剂
T75细胞培养瓶(Corning,目录号:CLS3275)
15 ml锥形管(Corning,目录号:CLS430791)
0.4 μ中号polyest ER的transwell插入物(VWR,目录号:734-1581)
聚赖氨酸涂层载玻片(Thermo Fisher Scientific,目录号:10219280)。
ARPE-19电池(ATCC,目录号:CRL-2302)
Alexa Fluor 488山羊抗兔(Thermo Fisher Scientific,目录号:A11034)
正常山羊血清(Sigma - Aldrich,目录号:G9023-10ml)
胶原IV(Sigma - Aldrich,目录号:C5533)
DMEM / F-12火腿(Sigma - Aldrich,目录号:D8437-500ML )
胎牛血清(Sigma - Aldrich,目录号:F9665-500ML )
青霉素链霉素(Sigma - Aldrich,目录号:P4333-100ML)
磷酸盐缓冲盐水(Sigma - Aldrich,目录号:D8662-500ML)
胰蛋白酶-EDTA(生物科学,目录号:25200-056)
正常人血清(Sigma - Aldrich,目录号:H4522-20ml)
ZO-1(Thermo Fisher Scientific,目录号:40-2200)
抗C5b-9(Santa Cruz Biotech,克隆ae11,目录号:sc58935)
Hoechst(Sigma - Aldrich,目录号:B2261-25MG)
MOWIOL ® 4-88 (西格玛- Aldrich公司,目录号:81381-50G)
甘油(Sigma-Aldrich,目录号:G5516)
Tris - H Cl (Thermo Fisher Scientific,目录号:10724344)
多聚甲醛(Sigma-Aldrich,目录号:158127)
NaOH (Sigma-Aldrich,目录号567530)
海卫一X-100(Sigma - Aldrich,目录号:x100-500ML)
L-谷氨酰胺(Thermo Fisher Scientific,目录号10378-016)
Mowiol 4-88(请参阅食谱)
PFA为4%(请参阅食谱)

设备


通风柜
血细胞计数器
-80°C冷冻室
4°C冰箱
镊子
生物安全柜ll级
热块
共焦激光扫描显微镜Axio Observer Z1倒置显微镜,配备Zeiss LSM 700 T-PMT扫描单元和40倍平面图。

程序


Transwell准备
外套0 0.4 μ米聚酯TR answell插入用100微克/ ml的在PBS中的胶原IV的吹打胶原蛋白的适当体积到的顶侧的transwell过滤器。
让transwell过滤器在细胞培养级生物安全柜中关闭盖子,使其风干。
从每个transwell过滤器中轻轻吸出其他胶原蛋白。
细胞接种前,将每个过滤器在PBS中清洗两次。

细胞接种ARPE-19
将ARPE-19细胞保存在T75细胞培养瓶中的DMEM / F-12 Ham中,其中含10%FBS和1%PS。细胞以80%汇合度传代(我们已使用它们直至传代30)。
从T75烧瓶中吸出培养基,并用5 ml无菌PBS洗涤细胞单层两次。
加入3 ml预热的胰蛋白酶并在37 °C下孵育5分钟,直到细胞开始分离。
加入3 ml DMEM F-12。
我们Ë 10 ml的stripette到吸6 ml的胰蛋白酶/媒体和冲洗T75的底壁以除去所有附着的细胞。
将细胞悬浮液转移到15 ml锥形管中。
205 × g离心5分钟沉淀细胞。
轻轻吸出媒体。
将细胞沉淀重悬于1 ml新鲜培养基中。
使用血细胞计数器计数细胞。
吸移管600 μ升的DMEM /含10个%FBS到的基底外侧面F-12火腿的transwell过滤器。
种子细胞以1.7的密度× 10 5细胞PE ř厘米2在上部的transwell在200室μ升的DMEM /含10个%FBS的F-12火腿,(transwell小表面积0.33厘米2每孔)。
两天后,用含1%FBS的DMEM / F-12更换介质。
每个星期,星期三和星期五更换介质4周(图2)。





图2 。在transwell滤膜上生长的RPE细胞的极化。A. RPE的示意图。极化的RPE细胞显示顶端微绒毛和ZO-1紧密连接蛋白表达。B.接种在跨膜过滤器的顶侧,并保持至少4周RPE细胞形成偏振光由下式定义ZO-1免疫组织化学在博所示单层日(C)ARPE-19细胞和(d)p rimary hfRPE细胞。比例尺= 20 µm 。


细胞接种原代人胎RPE(hfRPE )细胞
将细胞麻烦博士提供。来自美国贝塞斯达的美国国立眼科研究所(NEI)的Arvydas Maminishkis以融合的P-0单层形式被接收(Maminishkis等,2006)。
将hfRPE细胞放入装有培养基的T25中。
接受后,从细胞单层吸出所有培养基。
加入5 ml新鲜培养基Alpha MEM(不含L-谷氨酰胺Lonza BE12-169F),再加上Pen / strep + L-谷氨酰胺,5%FBS。
铺板前,让细胞至少静置1天。
吸出细胞培养液。
用50 ml的PBS洗涤细胞。
ñ OTE :牛逼他会充满整个瓶。


除去50 ml的PBS,重复进行第二步洗涤。
吸取5 ml温暖的胰蛋白酶-EDTA到烧瓶中。
将烧瓶放在培养箱中15分钟。
我们Ë脊椎穿刺针用3 ml的注射器,以吸出从RPE单层的胰蛋白酶。
将针的流动引导到带有粘附细胞的烧瓶壁上。
向下按压注射器以从烧瓶壁上流式清洗细胞。
我们Ë 5ml的吸量管向收集细胞悬浮液到无菌锥形管中。
立即将含有15%F BS的RPE培养基添加到锥形管中的细胞中。
再次重复步骤9-15,以确保所有细胞都被清除。
通过205 × g离心5分钟沉淀细胞。
轻轻吸出媒体。
将细胞沉淀重悬于1 ml新鲜培养基中(无需FCS)。
使用血细胞计数器计数细胞。
添加600 μ升RP的含有15个%FCS的每个的基底外侧孔E介质的transwell 。
种子细胞的密度100 × 10 3每厘米细胞2到心尖Transwell小在200室μ升含有15%FCS,RPE媒体的transwell小室表面积0.33厘米2每孔。
48小时后,从根尖和基底外侧transwell室抽吸培养基。
吸移管200 μ升含5个%FCS到顶端和600 RPE媒体的μ升到基底外侧室。
继续每个星期一,星期三和星期五更换RPE媒体4周。

验证紧密连接的形成
注意:紧密连接形成的验证可以与MAC形成分析同时进行。


4周后在培养的RPE细胞上的transwell插入物加入200固定μ升4%低聚甲醛(PFA)的至顶膜和600 μ升4%PFA的至基底外侧室。
在室温下将细胞在PFA中孵育1 0分钟。
轻轻抽吸PFA,注意不要在Transwell过滤器上戳孔或扰乱细胞。
洗涤细胞在过滤器上5分钟3次用200 μ升在顶部隔室的PBS和600 μ升的基底外侧面。
吸移管200 μ升或600 μ升0.05%的Triton X - 100分别插入到顶端和基底外侧腔室。
将细胞在室温下于0.05%Triton X - 100中孵育10分钟,以透化细胞膜。
吸取Triton X - 100。
吸移管200 μ升或600 μ升5%正常山羊血清(NGS)的在所述顶端和基底外侧分别室。
在室温下在NGS中封闭细胞1小时。
吸出5%NGS。
吸移管100 μ升ZO-1抗体(1:100 5%NGS)插入的顶侧的transwell过滤器。
100 μ升抗体的移液到一个空6的底部-孔板和所述的transwell插入物被直接放置在抗体坐。
将插入物在4 °C下孵育过夜。
从心尖腔抽吸抗体。
地方的transwell与600一个新板μ升在基底外侧腔和吸移管100的PBS μ升PBS进入顶侧。
重复此洗涤步骤3次。
吸移管100 μ升或600 μ升PBS与山羊抗-兔488(1:500)到顶端和基底外侧分别室。
孵育转孔中的第二抗体2 ħ一吨室温。
吸出二抗。
吸移管200 μ升或600 μ升分别PBS进入顶端和基底外侧腔室。
重复PBS洗涤3次。
吸出最后的PBS
加入200 μ升的Hoechst的(1:10 ,000)到心尖的transwell 。
孵育Transwell小为2分钟。
在PBS中将transwell洗涤3次。
轻轻地从Transwell过滤器中清除所有液体。
将transwell倒置在干净的表面上。
ģ ently ü小号Ë无菌手术刀来切割绕膜的周边上。
第一次切开后,用无菌镊子夹住过滤器的一侧。
继续用刀片缓慢切割时,请轻轻用镊子松开膜。注意不要用镊子刮擦细胞单层。
一旦膜被切开,用镊子将膜的基底外侧向下安装在聚赖氨酸涂层的玻片上。即,单元格应面向幻灯片顶部。
吸管50-100 μ升的的Mowiol ® 4-88安装媒体到膜上并与盖盖玻片。
MAC形成分析
在MAC形成分析之前48小时,从根尖和基底外侧transwell室中取出所有RPE培养基。
吸移管600 μ升无血清的DMEM / F-12火腿到基底外侧transwell小室中。
吸移管200 μ升无血清的DMEM / F-12火腿入到顶室。
在37°C时快速解冻普通人血清(NHS)的瓶装。
等分试样NHS成单次使用的等分试样(300-500 μ升)。
将等分的NHS储存在-80°C。
在MAC分析的当天,从-80°C除去两等分的NHS。
在冰上解冻等分试样。
通过放置在设定为56°C的加热块或水浴中,加热可灭活一份等量的NHS。
在56°C下孵育血清30分钟,每10分钟倒转一次试管。
让热灭活的血清冷却至室温。
指定哪些transwell将用于顶端或基底外侧的MAC形成。
从Transwell抽吸培养基。
添加600 μ升无血清培养基中的至基底外侧室和180 μ升无血清培养基中的被指定为心尖MAC评估井的顶室中。
添加540 μ升无血清培养基中的至基底外侧室和200 μ升无血清培养基中,以指定用于基底外侧MAC评估井的顶室中。
移液管20 μ升任NHS或热灭活(您好)HIN HS到含有180孔的顶室μ升的培养基。
移液管60 μ升任NHS或HiNHS到含有540孔的基底外侧室μ升的培养基。
将RPE与NHS或HiNHS孵育2-24小时。
后通过添加200 24个小时修复细胞μ升4%PFA的至顶膜和600 μ升4%PFA基底外侧室。
在室温下将细胞在PFA中孵育10分钟。
轻轻抽吸PFA,注意不要在Transwell过滤器上戳孔或干扰细胞。
洗涤细胞在过滤器上5分钟用200 3次μ升在顶部隔室的PBS和600 μ升的基底外侧面。
吸移管200 μ升或600 μ升血清白蛋白(BSA)到顶端和基底外侧腔室分别牛的5%。
在室温下在BSA中封闭细胞1小时。
吸取5%BSA。
吸移管100 μ升的C5b-9 MAC抗体(在5 1:25%BSA)到API的所述的卡侧的transwell过滤器。
100 μ升抗体的移液到一个空6的底部-孔板和所述的transwell插入物被直接放置在抗体坐,板带盖并用石蜡膜密封。
将插入物在冰箱中于4°C孵育过夜。
从心尖腔抽吸抗体。
地方的transwell与600一个新板μ升在基底外侧腔和吸移管100的PBS μ升PBS进入顶侧。
重复此洗涤步骤3次。
吸移管100 μ升或600 μ升PBS与山羊抗小鼠647(1:500)和鬼笔环肽(1:500)到顶端和基底外侧分别室。
孵育转孔中的第二抗体2 ħ一吨室温。
吸出二抗。
吸移管200 μ升或600 μ升分别PBS进入顶端和基底外侧腔室。
重复PBS洗了3次。
吸出最后的PBS
加入200 μ升的Hoechst的(1:10 ,000)到心尖的transwell 。
孵育Transwell 2分钟。
在PBS中将transwell洗涤3次。
轻轻地从Transwell过滤器中清除所有液体。
将transwell倒置在干净的表面上。
ģ ently使用无菌手术刀来切割绕膜的周边上。
第一次切开后,用无菌镊子夹住过滤器的一侧。
继续用刀片缓慢切割时,请轻轻用镊子松开膜。注意不要用镊子刮擦细胞单层。
一旦膜被切开,使用镊子将膜安装到聚赖氨酸包被的玻片上(Thermo Scientific)。
吸管50-100 μ升的的Mowiol  4-88安装媒体到膜上并与盖盖玻片。
使用共聚焦激光扫描显微镜对ARPE-19细胞(图3)和hfRPE细胞(图4)上的MAC形成进行成像。





图3 。在ARPE-19细胞上进行MAC分析。在CEP刺激的ARPE-19细胞中(诱导替代补体途径)和10%NHS作为补体来源24小时观察到MAC形成(红色)。在单独的NHS或H iNHS对照中均未观察到染色。比例尺= 20 μ米。






图4在hfRPE上的MAC分析。在用CEP和10%NHS刺激2小时的hfRPE细胞中观察到MAC形成(绿色)。仅在NHS或HiNHS对照中未观察到染色。比例尺= 20 μ米。






数据分析


使用配有Zeiss LSM 700 T-PMT扫描单元和40倍平面图的共聚焦激光扫描显微镜Axio Observer Z1倒置显微镜分析染色。


菜谱


Mowiol 4-88
2 .4克Mowiol


6克甘油


6毫升H 2 O


在室温下搅拌几个小时


加入12 ml 0.2 M Tris -H Cl(pH 8.5)并加热至50 °C 10分钟


离心机的Mowiol在5 ,000 ×克15分钟


分装并储存在-20°C


PFA 4%
将4 g PFA添加到50 ml H 2 O中
加入1 ml 1 M NaOH,在约60°C的加热块上轻轻搅拌直至PFA溶解
加入10毫升10 × PBS,让混合物冷却至室温
调节pH至7.4
用H 2 O调节最终体积至100 ml
通过0.45-μm的膜滤器过滤溶液以除去任何颗粒物
使PFA溶液在使用前新鲜,或等分保存在−20°C几个月
注意小号:


避免反复冻结/解冻。
制备PFA会释放出有毒烟雾,我们使用通风橱作为预防措施。

致谢


这项研究由美国BrightFocus基金会(M2016030 ),爱尔兰卫生研究委员会(HRB-HRA / 2013.290 )和爱尔兰科学基金会(SFI 15 / CDA / 3497 ),皇家维多利亚州眼耳医院(RVEEH)资助,爱尔兰,国家儿童研究中心(NCRC),爱尔兰,爱尔兰研究委员会(IRCLA / 2017/295 ),爱尔兰。显示该方案的原始研究论文发表在细胞报告中(Mulfaul et al。,2020)。


利益争夺


作者没有金融或非金融竞争利益。


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引用:Mulfaul, K. and Doyle, S. (2021). In vitro Measurement of Membrane Attack Complex in RPE Cells. Bio-protocol 11(4): e3916. DOI: 10.21769/BioProtoc.3916.
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