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Mouse Model of Immune Complex-mediated Vasculitis in Dorsal Skin and Assessment of the Neutrophil-mediated Tissue Damage
免疫复合物介导的小鼠背部皮肤血管炎模型及中性粒细胞介导的组织损伤评估   

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Stem Cells
Sep 2016

Abstract

Neutrophils are the most abundant leukocytes in the blood. In the recent decades, their crucial roles in host defense, immune regulation and tissue damage have been studied in a deeper dimension. In this protocol, we described a mouse model of immune complex-mediated vasculitis in the dorsal skin induced by Arthus reaction, and the subsequent analysis of edema, hemorrhage and tissue damage due to neutrophil activation by means of Evans blue area analysis, histology, and immunofluorescence. This protocol could facilitate the investigation of cellular therapy strategy against over-activated neutrophil-mediated tissue damage.

Keywords: Vasculitis (血管炎), Neutrophil (中性粒细胞), Evans blue (伊文思蓝), Tissue damage (软组织损伤)

Background

Neutrophils constitute the largest, evolutionary conserved fraction of circulating leukocytes. They lead the first wave of host defense against infection or tissue damage. In vitro models of neutrophil-mediated cellular cytotoxicity are well established (Incani et al., 1981; Dallegri et al., 1984; Saffarzadeh et al., 2012). However, to dissect the complexity of neutrophil-mediated sterile tissue injury, in vivo models are indispensable.

Immune complex (IC)-mediated vasculitis is a disease initiated by the deposition of antigen-antibody complexes in blood vessels, which subsequently lead to complement activation, neutrophil recruitment and activation. The large amount of reactive oxygen species and proteases released from activated neutrophils damage the endothelial lining of the vessel wall and result in edema and hemorrhage (Sindrilaru et al., 2007; Goerge et al., 2008; Feld et al., 2012). The previously described IC-mediated vasculitis induced by Arthus reaction in mouse ears (Sindrilaru et al., 2007) is one of successful models to study the neutrophil-mediated tissue damage. However, with very thin layer of tissue, mouse ear is not suitable to study the effect of potential therapeutic agents in a large volume, for example, with cellular therapy strategy.

In this protocol, we describe a mouse model of IC-mediated vasculitis in dorsal skin, which enables to overcome the above-mentioned pitfall. Furthermore, we provide the details of subsequent analysis of neutrophil induced hemorrhage and tissue injury by Evans blue quantification, together with histological and immunofluorescence techniques. Our results indicated that this mouse model closely resembles the features of tissue damage in human vasculitis patients caused by over-activated neutrophils. This protocol has been applied successfully for our recent discoveries that mesenchymal stem cells suppress hemorrhage and tissue damage in immune-complex mediated vasculitis mediated by over-activated neutrophils (Jiang et al., 2016).

Materials and Reagents

  1. 1 ml syringe (Omnifix-F) (B. Braun Medical, catalog number: 9161406V-02 )
  2. Needle 26 G x ½” (B. Braun Medical, catalog number: 4665457-02 )
  3. 0.22 µm filter (Merck, catalog number: SLGV033RS )
  4. Cover glasses, 24 x 55 mm, thickness No. 1 (VWR, catalog number: 631-0146 )
  5. C57BL/6J mice at preferred age of 8-12 weeks, both male and female are suitable for this protocol (THE JACKSON LABORATORY, catalog number: 000664 )
  6. Anti-bovine albumin antibody produced in rabbit, whole antiserum (Sigma-Aldrich, catalog number: B1520 ) (aliquots store at -20 °C)
  7. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
  8. Formalin solution, neutral buffered, 10% (Sigma-Aldrich, catalog number: HT5014 )
  9. Xylene (Carl Roth, catalog number: CN80.1 )
  10. Ethanol (Carl Roth, catalog number: 9065.2 )
  11. Hematoxylin solution acid acc. to Mayer (Carl Roth, catalog number: T865 )
  12. Eosin Y solution 0.5% in water (Carl Roth, catalog number: X883 )
  13. Acetic acid (Carl Roth, catalog number: 3738.1 )
  14. Roti-histol (Carl Roth, catalog number: 6640 )
  15. Roti-histokitt (Carl Roth, catalog number: 6638 )
  16. Target retrieval solution, 10x (Agilent Technologies, DAKO, catalog number: S169984-2 )
  17. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  18. Purified rabbit anti-human/mouse neutrophil elastase/NE antibody (Abcam, catalog number: ab68672 )
  19. Purified rat anti-mouse Ly6G antibody (Clone RB6-8C5) (Abcam, catalog number: ab25377 )
  20. Alexa Fluor 488-conjugated goat anti-rat IgG secondary antibody (Thermo Fisher Scientific, Invitrogen, catalog number: A-11006 )
  21. Alexa Fluor 488-conjugated donkey anti-rabbit IgG secondary antibody (Thermo Fisher Scientific, Invitrogen, catalog number: A-21206 )
  22. Purified mouse IgG2a κ isotype control (clone C1.18.4) (BD, BD Biosciences, catalog number: 550339 )
  23. Purified mouse anti-DNA/histone H1 antibody (Merck, catalog number: MAB3864 )
  24. Purified goat anti-human/mouse myeloperoxidase/MPO antibody (R&D Systems, catalog number: AF3667 )
  25. Alexa Fluor 555-conjugated goat anti-mouse IgG secondary antibody (Thermo Fisher Scientific, Invitrogen, catalog number: A-21422 )
  26. Alexa Fluor 555-conjugated donkey anti-goat IgG secondary antibody (Thermo Fisher Scientific, Invitrogen, catalog number: A-21432 )
  27. 4’,6-Diamidino-2-phenylindole, Dihydrochloride (DAPI) (Thermo Fisher Scientific, InvitrogenTM, catalog number: D1306 )
  28. Fluorescence mounting medium (Agilent Technologies, DAKO, catalog number: S302380-2 )
  29. Ketanest S 25 mg/ml (ketamine) (Pfizer, authorization number: 39945.00.00)
  30. Rompun 2% (xylazine) (Bayer HealthCare, drug identification number: 02169606 )
  31. Evans blue (Sigma-Aldrich, catalog number: E2129 )
  32. Rabbit serum (Sigma-Aldrich, catalog number: R9133 )
  33. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  34. Goat serum (Sigma-Aldrich, catalog number: G9023 )
  35. Fetal bovine serum (FBS) (Biochrom, catalog number: S 0615 )
  36. Anesthesia solution (see Recipe 1)
  37. Evans blue-BSA solution (see Recipe 2)
  38. Blocking buffer (see Recipe 3)
  39. Antibody diluent (see Recipe 4)

Equipment

  1. Balance
  2. Heating table (e.g., MEDAX, model: 13801 )
  3. Animal hair clipper (e.g., Aesculap Exacta Tierschermaschine, B. Braun Medical, Aesculap, catalog number: GT415 )
  4. Tissue processor (e.g., Leica Biosystems, model: Leica TP1020 )
  5. Digital camera (e.g., Panasonic, model: Lumix DMC-LX7 )
  6. Fluorescent microscope: Zeiss Axiophot microscope with an AxioCam digital color camera and AxioVision software v4.7 (Carl Zeiss)
  7. Steam cooker (e.g., Braun MultiGourmet Food Steamer, Braun, model: FS 20 )
  8. Slide staining trays (e.g., StainTrayTM Systems for 20 Slides with Base with Black Cover, Simport, catalog number: M920-2 )

Software

  1. ImageJ
  2. Graphpad Prism
  3. AxioVision (ZEISS)

Procedure

  1. Arthus reaction in dorsal skin
    1. Weigh the experimental C57BL/6J mice and intraperitoneally inject anesthesia solution (see Recipe 1) according to the body weight at 5 µl/g body weight (e.g., 150 µl for a 30 g mouse). The mice are expected to be anesthetized for at least 30 min. Mice are placed on a heating table (39 °C) during anesthesia to maintain the body temperature.
    2. Shave the hair on the dorsal skin by using an animal hair clipper.
    3. Optional: application of therapeutic agents and the respective controls.
      Notes:
      1. The method and duration of application of therapeutic agents need to be determined for individual treatment condition. For example, to study the effect of mesenchymal stem cells (MSCs) on IC-mediated vasculitis, 2.5 x 105 adipose tissue-derived MSCs in 100 µl PBS per injection were intradermally injected to both sides of the shaved dorsal skin of mice.
      2. Mice intradermally injected with 100 µl PBS served as controls. Injection of such volumes into mouse ears is not possible.
      3. Arthus reaction can be induced half an hour after injection of MSCs.
    4. Intravenously (or intraperitoneally) inject 100 µl of Evans blue-BSA solution (see Recipe 2).
      Note: Compared to i.v. injection, i.p. injection resulted in smaller Evans blue areas in the skin at the injection sites of anti-BSA antibody, but the difference between anti-BSA and rabbit serum control was clear (Figure S1). Therefore, i.p. injection of Evans blue-BSA solution can also be used for this procedure.
    5. Intradermally inject 40 µl of rabbit anti-BSA antibody (undiluted whole antiserum, concentration varies in different lots, in our experiments ranged from 3-5 mg/ml) into the dorsal skin of interest (e.g., areas that had been treated testing therapeutic agent, such as with AT-MSCs, or respective control). In control mice, 40 µl of rabbit serum was injected intradermally.
    6. Four hours later, to quantify the extent of Evans blue-marked vascular leakage, sacrifice the mice, harvest skin specimen (Video 1) and immediately photograph digitally (Figure 1B).

      Video 1. Dissection of mouse skin after Arthus reaction. The Arthus reaction was elicited by i.p. injection of 100 µl PBS solution containing 1% Evans blue and 2% BSA. Afterward, 40 µl of anti-BSA antibody (left-side of back-skin) or 40 µl rabbit serum (right-side of back-skin) was intradermally injected. 4 h later, mice were sacrificed and the skin specimen was harvested and digitally photographed.

    7. Analyze the Evans blue areas in the skin, indicative of the extent of vessel damage due to BSA/anti-BSA complex formation, with ImageJ (details see Data analysis) (Figure 1).


      Figure 1. Induction of IC-mediated vasculitis in mouse dorsal skin and data analysis. A. Flow chart of the data analysis; B. 100 µl PBS or 2.5 x 105 adipose tissue-derived mesenchymal stem cells (MSCs) were intradermally injected to both sides of the shaved dorsal skin of mice. The Arthus reaction was elicited by i.v. injection of 100 µl PBS solution containing 1% Evans blue and 2% BSA. Afterward, 40 µl of anti-BSA antibody was intradermally injected to the area that had been injected with MSCs or PBS. 4 h later, mice were sacrificed and the skin specimen was harvested and digitally photographed. C. With ImageJ, the original photo in RGB format was converted to HSB format with plugin ‘Color Space Converter’. D. After splitting channels, the green channel was kept for Evans blue area analysis. D’. Alternatively, the RGB photo was converted to CYMK format with plugin ‘RGB to CYMK’ and the cyan channel was kept for analysis. E. A binary image was created with the Evans blue areas in black. F and G. Settings for measurement and particle analysis. H. Particle analysis indicating the Evans blue areas in skin. I. Results were presented as a scatter plot by GraphPad Prism. Intradermal injection of MSCs resulted in significantly reduced areas of Evans blue staining by 52.1% compared to PBS control (15.8 vs. 33.0 AU). **, P < 0.01 by Mann-Whitney tests; AU, arbitrary unit.

  2. Histology and immunofluorescence of neutrophils
    Note: Histological examination with H&E staining and immunofluorescence staining of neutrophil activation markers are optional but are recommended, in order to confirm the observed tissue damage is indeed due to neutrophil recruitment and activation. 
    1. Arthus reaction without Evans blue
      Follow the Procedure A. In Step A4, replace the Evans blue-BSA solution to sterile filtered 2% BSA in PBS. In Step A6, the harvested skin specimen was fixed in 10% formalin solution at 4 °C for overnight. Fixed tissues were processed with tissue processor for dehydration and clearing and embedded in paraffin blocks and sectioned into 5 µm sections using a microtome (the details of making formalin-fixed-paraffin-embedded [FFPE] sections can be referred to existing literature, e.g., Canene-Adams, 2013).
    2. H&E staining (Figure 2)
      1. Heat paraffin sections at 50-60 °C for 30 min.
      2. Put sections in xylene for 10 min for 2 times.
      3. Hydrate sections in 100%, 95%, 80%, 70% ethanol for 3 min for 2 times for each.
      4. Incubate the sections in distilled water for 5 min.
      5. Incubate in Mayer’s hematoxylin for 10 min.
      6. Wash in cold tap water for 5 min.
      7. Rinse with distilled water (dip the slides in distilled water for several times).
      8. Incubate in Eosin Y (+ 1 drop of acetic acid/100 ml) for 2 min.
      9. Rinse with distilled water.
      10. Dehydrate the sections in 80%, 95%, 100% ethanol for 5 min each.
      11. Clear with Roti-histol.
      12. Mount with Roti-histokitt.


        Figure 2. H&E staining of FFPE skin sections after induction of IC-mediated vasculitis. Mice were intradermally injected with PBS or MSCs and subjected to Arthus reaction by i.v. injection of 100 µl PBS solution containing 2% BSA. Afterwards, anti-BSA antibody was intradermally injected into the area that had been injected with MSCs or PBS. Mice received rabbit serum served as healthy controls. 4 h later, mice were sacrificed and the skin specimen was harvested and processed to 5 µm paraffin-embedded sections. Representative pictures of H&E staining of skin sections from 4 mice of each group are shown. White arrows indicate blood vessels. Scale bars = 50 µm. The dermis of the mice subjected to Arthus reaction with PBS injection showed severe damage of vessels surrounded by red blood cells and neutrophils (B). Pre-treatment of MSCs reduced vessel destruction (C), and histology was similar to skin specimen from healthy control mice (A).

    3. Immunofluorescence staining of neutrophil marker (Ly6G), neutrophil extracellular traps (NETs), neutrophil elastase (NE) and myeloperoxidase (MPO)
      1. Follow Steps B2a-B2d.
      2. During Step B3a, prepare antigen retrieval buffer: 20 ml of 10x target retrieval solution + 180 ml distilled water, and heat the buffer in a steam cooker.
      3. Antigen retrieval: heat sections (in plastic chambers) in buffer for 15 min in a steam cooker.
      4. Cooling for 10 min, and put sections in PBS for 5 min.
      5. Permeabilization: put sections in 0.25% Triton X-100 for 10 min (0.5 ml Triton X-100 in 200 ml PBS).
      6. Wash with PBS for 3 times with each 5 min.
      7. Incubate the sections with blocking buffer (see Recipe 3) for 1 h at room temperature.
      8. Incubate the sections with the first primary antibody at 4 °C for overnight in a staining tray: anti-Ly6G (1:100 dilution in antibody diluent [see Recipe 4]) (Figures 3A-3C), or anti-NE (1:200 dilution in antibody diluent) (Figures 3D-3F).
      9. Wash in PBS 3 times, each time 5 min.
      10. Incubate the section with the first secondary antibody at room temperature for 1 h in a staining tray: AF488-conjugated goat anti-rat IgG (1:200 dilution in antibody diluent) (Figures 3A-3C), or AF488-conjugated donkey anti-rabbit IgG (1:200 dilution in antibody diluent) (Figures 3D-3F).
      11. Wash in PBS 3 times, each time 5 min. Avoid the direct light exposure.
      12. Incubate the section with the second primary antibody at room temperature for 2 h in a staining tray: anti-DNA/histone H1 antibody for NETs (1:100 dilution in antibody diluent) (Figures 3A-3C), or anti-MPO (1:50 dilution in antibody diluent) (Figures 3D-3F).
      13. Wash in PBS 3 times, each time 5 min.
      14. Incubate the section with the second secondary antibody at room temperature for 1 h in a staining tray: AF555-conjugated goat anti-mouse IgG (1:200 dilution in antibody diluent) (Figures 3A-3C), or AF555-conjugated donkey anti-goat IgG (1:200 dilution in antibody diluent) (Figures 3D-3F).
      15. Wash in PBS 3 times, each time 5 min. Avoid the direct light exposure.
      16. Incubate the section with DAPI solution (1:5,000 dilution in PBS) at room temperature for 3 min in a staining tray.
      17. Rinse the slides with PBS, and air dry. Avoid the direct light exposure.
      18. Mount cover slip with fluorescent mounting media. The slides can be stored at 4 °C.
      19. Fluorescence microscopy.


        Figure 3. Immunofluorescence staining of neutrophil activation markers on FFPE skin sections after induction of IC-mediated vasculitis. Mice were intradermally injected with PBS or MSCs and subjected to Arthus reaction by i.v. injection of 100 µl PBS solution containing 2% BSA. Afterwards anti-BSA antibody was intradermally injected into the area that had been injected with MSCs or PBS. Mice received rabbit IgG served as healthy controls. 4 h later, mice were sacrificed and the skin specimen was harvested and processed to 5 µm paraffin-embedded sections. Representative pictures of skin sections from 4 mice of each group with immunostaining of murine neutrophil marker Ly6G (green) and NETs marker DNA/histone-1 (red) (A-C), and immunostaining of NE (green) and MPO (red) (D-F) are shown. Nuclei were counterstained with DAPI (blue) in immunostainings. Scale bars = 50 µm. The dermis of the mice subjected to Arthus reaction with PBS injection showed massive infiltration of neutrophils with NETs (B), NE and MPO in the tissue (E), which very much resemble the features of human vasculitis. In contrast, intradermal injection of MSCs significantly reduced NETs (C) and the numbers of NE+MPO+ neutrophils in the dermis (F), which were similar to skin specimen from healthy control mice (A and D).

Data analysis

Analysis of areas of Evans blue in skin affected by Arthus reaction by ImageJ. The flow chart of the analysis is shown in Figure 1A.

  1. Download ImageJ plugin ‘Color Space Converter’ under the category ‘Color’:
    https://imagej.nih.gov/ij/plugins/color-space-converter.html. Save ‘Color_Space_Converter.jar’ in the plugins folder or subfolder, e.g., Tools: ...\ImageJ\plugins\Tools
  2. Open the original RGB photo. ‘Plugins-Tools-Color Space Converter’: Choose RGB to HSB, leave other settings alone, click ‘OK’ (Figure 1C).
  3. ‘Image-Color-Split channels’. Use the green channel for the area analysis (Figure 1D), close the other 2 channels (red and blue).
    Note: Alternative Steps 1-3; alternative plugin to be used: ‘RGB to CYMK’.
    Download the plug-in at https://imagej.nih.gov/ij/plugins/cmyk/index.html. After channel split, keep the cyan (C) channel (Figure 1D’) to convert to binary, close other channels (M, Y, K).
  4. ‘Image-Adjust-Threshold’. Check ‘dark background’ and set the threshold to a fixed value for all the photos and apply. This creates a binary (black and white) image with the Evans blue areas in black (Figure 1E). Set the background value to have a balance in adequate intensity of Evans blue signal but minimized background.
  5. On the binary, ‘Analyze-set measurements’: check ‘Area’, leave other options alone, click ‘OK’ (Figure 1F).
  6. ‘Analyze-analyze particles’: Set ‘size (pixel^2)’: e.g., 2000-infinity. ‘Show’: choose ‘overlay’.
    Check ‘Display results’ (Figures 1G and 1H) (the size value may need to adjust for smaller areas, depends on what is counted in the end, details see Notes section). You may need manually remove the data of non-Evans blue background areas.
  7. Save the results in GraphPad Prism for analysis. This assay is recommended to be performed with at least 5 mice per treatment group. Data could be presented as a scatter plot (vertical) by GraphPad Prism. Mann-Whitney tests can be used for significance test (Figure 1I).

Notes

  1. Troubleshooting for ImageJ data analysis
    1. If you do not see a window with results opening, you forgot to check ‘Display results’ in ‘Analyze–analyze particles’.
    2. If you do not see which number of area corresponds to which area in the binary, you forgot to set ‘Show’ to ‘overlay’ in ‘Analyze–analyze particles’.
    3. If you get hundreds of very small areas, you should increase the minimum size of particles in ‘Analyze–analyze particles’.
    4. If one of your black areas is not analyzed, you should increase minimum size of particles in ‘Analyze–analyze particles’.

Recipes

  1. Anesthesia solution (ketamine and xylazine based)
    Per 10 ml anesthesia solution contains:
    4 ml of Ketanest S 25 mg/ml
    0.5 ml of 2% rompun
    5.5 ml of sterile PB
  2. Evans blue-BSA solution (can be kept at 4 °C for a week)
    1% Evans blue + 2% BSA in PBS, pass through a 0.22 µm filter
  3. Blocking buffer (prepare fresh)
    PBS containing 5% BSA + 5% goat serum in PBS
  4. Antibody diluent (prepare fresh)
    PBS containing 1% BSA

Acknowledgments

This work was supported in part by research grants from the Baden-Württemberg Stiftung (P-BWS-ASII/15), the European Commission (CASCADE HEALTH-FP7-223236) and the German Research Foundation (SFB1149) to K.S.-K., the Baustein Program from the Medical Faculty, University of Ulm (LSBN.0100) to D.J. Part of figures are adapted and modified from the study of Jiang et al., 2016. The authors indicate no potential conflicts of interest.

References

  1. Canene-Adams, K. (2013). Preparation of formalin-fixed paraffin-embedded tissue for immunohistochemistry. Methods Enzymol 533: 225-233.
  2. Dallegri, F., Patrone, F., Frumento, G., Ballestrero, A. and Sacchetti, C. (1984). Neutrophil-mediated cellular cytotoxicity triggered by immobilized aggregated IgG: an in vitro model of cell injury during immune complex diseases. J Clin Immunol 4(6): 439-444.
  3. Feld, M., Goerge, T., Hillgruber, C., Steingraber, A. K., Fastrich, M., Shpacovitch, V. and Steinhoff, M. (2012). α-1-antitrypsin and IFN-γ reduce the severity of IC-mediated vasculitis by regulation of leukocyte recruitment in vivo. J Invest Dermatol 132(9): 2286-2295.
  4. Goerge, T., Ho-Tin-Noe, B., Carbo, C., Benarafa, C., Remold-O’Donnell, E., Zhao, B. Q., Cifuni, S. M. and Wagner, D. D. (2008). Inflammation induces hemorrhage in thrombocytopenia. Blood 111(10): 4958-4964.
  5. Incani, R. N. and McLaren, D. J. (1981). Neutrophil-mediated cytotoxicity to schistosomula of Schistosoma mansoni in vitro: studies on the kinetics of complement and/or antibody-dependent adherence and killing. Parasite Immunol 3(2): 107-126.
  6. Jiang, D., Muschhammer, J., Qi, Y., Kugler, A., de Vries, J. C., Saffarzadeh, M., Sindrilaru, A., Beken, S. V., Wlaschek, M., Kluth, M. A., Ganss, C., Frank, N. Y., Frank, M. H., Preissner, K. T. and Scharffetter-Kochanek, K. (2016). Suppression of neutrophil-mediated tissue damage-a novel skill of mesenchymal stem cells. Stem Cells 34(9): 2393-2406.
  7. Saffarzadeh, M., Juenemann, C., Queisser, M. A., Lochnit, G., Barreto, G., Galuska, S. P., Lohmeyer, J. and Preissner, K. T. (2012). Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. PLoS One 7(2): e32366.
  8. Sindrilaru, A., Seeliger, S., Ehrchen, J. M., Peters, T., Roth, J., Scharffetter-Kochanek, K. and Sunderkotter, C. H. (2007). Site of blood vessel damage and relevance of CD18 in a murine model of immune complex-mediated vasculitis. J Invest Dermatol 127(2): 447-454.

简介

嗜中性粒细胞是血液中最丰富的白细胞。 近几十年来,它们在宿主防御,免疫调节和组织损伤方面的关键作用已经得到了更深入的研究。 在这个协议中,我们描述了Arthus反应诱导的背部皮肤中的免疫复合物介导的血管炎的小鼠模型,以及随后通过伊文思蓝区域分析,组织学和分析由于嗜中性粒细胞活化引起的水肿,出血和组织损伤免疫荧光。 该协议可以促进细胞治疗策略对过度活化的嗜中性粒细胞介导的组织损伤的调查。

【背景】嗜中性粒细胞构成循环白细胞的最大的进化保守部分。他们引导第一波主机防御感染或组织损伤。嗜中性粒细胞介导的细胞毒性的体外模型已被充分证实(Incani等人,1981; Dallegri等人,1984; Saffarzadeh等人,等人,2012年)。但是,为了剖析中性粒细胞介导的无菌组织损伤的复杂性,“体内”模型是必不可少的。

免疫复合物(IC)介导的血管炎是由抗原 - 抗体复合物在血管中沉积引发的疾病,其随后导致补体激活,嗜中性粒细胞募集和活化。活化的嗜中性粒细胞释放的大量活性氧和蛋白酶损害血管壁的内皮衬里并导致水肿和出血(Sindrilaru等人,2007; Goerge等人, ,2008; Feld等人,2012)。之前描述的由Arthus反应在小鼠耳中诱导的IC介导的血管炎(Sindrilaru等人,2007)是研究嗜中性粒细胞介导的组织损伤的成功模型之一。然而,对于非常薄的组织层,小鼠耳朵不适合大量研究潜在治疗剂的效果,例如,用细胞治疗策略。

在这个协议中,我们描述了背部皮肤的IC介导的血管炎的小鼠模型,其使得能够克服上述的缺陷。此外,我们提供了伊文思蓝定量随后分析中性粒细胞诱导的出血和组织损伤的细节,以及组织学和免疫荧光技术。我们的研究结果表明,这个小鼠模型非常类似于过度活化的嗜中性粒细胞引起的人血管炎患者的组织损伤的特征。该方案已成功应用于我们最近发现的间充质干细胞抑制过度活化的嗜中性粒细胞介导的免疫复合物介导的血管炎中的出血和组织损伤(Jiang等人,2016)。

关键字:血管炎, 中性粒细胞, 伊文思蓝, 软组织损伤

材料和试剂

  1. 1ml注射器(Omnifix-F)(B.Braun Medical,目录号:9161406V-02)
  2. 针26 G x½“(B. Braun医疗,目录号:4665457-02)
  3. 0.22μm过滤器(Merck,目录号:SLGV033RS)
  4. 覆盖眼镜,24×55毫米,厚度1号(VWR,目录号:631-0146)
  5. C57BL / 6J小鼠,优选年龄为8-12周,男性和女性都适合本方案(THE JACKSON LABORATORY,目录号:000664)
  6. 在兔子中产生的抗牛血清白蛋白抗体,全抗血清(Sigma-Aldrich,目录号:B1520)(等分试样储存在-20℃)
  7. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190144)
  8. 福尔马林溶液,中性缓冲液,10%(Sigma-Aldrich,目录号:HT5014)
  9. 二甲苯(卡尔罗斯,目录号:CN80.1)
  10. 乙醇(卡尔罗斯,目录号:9065.2)
  11. 苏木素溶液酸迈尔(卡尔罗斯,目录号:T865)
  12. 在水中的曙红Y溶液0.5%(Carl Roth,目录号:X883)
  13. 乙酸(卡尔罗斯,目录号:3738.1)
  14. Roti-histol(Carl Roth,目录号:6640)
  15. Roti-histokitt(Carl Roth,目录号:6638)
  16. 目标检索解决方案,10倍(安捷伦科技公司,DAKO,目录号:S169984-2)
  17. Triton X-100(Sigma-Aldrich,目录号:X100)
  18. 纯化的兔抗人/小鼠嗜中性粒细胞弹性蛋白酶/ NE抗体(Abcam,目录号:ab68672)
  19. 纯化的大鼠抗小鼠Ly6G抗体(克隆RB6-8C5)(Abcam,目录号:ab25377)
  20. Alexa Fluor 488-结合的山羊抗大鼠IgG二抗(Thermo Fisher Scientific,Invitrogen,目录号:A-11006)
  21. Alexa Fluor 488-偶联的驴抗 - 兔IgG二抗(Thermo Fisher Scientific,Invitrogen,目录号:A-21206)
  22. 纯化的小鼠IgG2aκ同种型对照(克隆C1.18.4)(BD,BD Biosciences,目录号:550339)
  23. 纯化的小鼠抗DNA /组蛋白H1抗体(Merck,目录号:MAB3864)
  24. 纯化的山羊抗人/小鼠髓过氧化物酶/ MPO抗体(R&amp; D Systems,目录号:AF3667)
  25. Alexa Fluor 555偶联山羊抗小鼠IgG二抗(Thermo Fisher Scientific,Invitrogen,目录号:A-21422)
  26. Alexa Fluor 555偶联的驴抗山羊IgG二抗(Thermo Fisher Scientific,Invitrogen,目录号:A-21432)
  27. 4',6-二脒基-2-苯基吲哚二盐酸盐(DAPI)(Thermo Fisher Scientific,Invitrogen TM,目录号:D1306)
  28. 荧光固定介质(Agilent Technologies,DAKO,目录号:S302380-2)
  29. Ketanest S 25 mg / ml(氯胺酮)(辉瑞公司,授权号:39945.00.00)
  30. Rompun 2%(甲苯噻嗪)(Bayer HealthCare,药物识别号码:02169606)
  31. 伊文思蓝(Sigma-Aldrich,目录号:E2129)
  32. 兔血清(Sigma-Aldrich,目录号:R9133)
  33. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906)
  34. 山羊血清(Sigma-Aldrich,目录号:G9023)
  35. 胎牛血清(FBS)(Biochrom,目录号:S 0615)
  36. 麻醉解决方案(见配方1)
  37. 伊文思蓝BSA解决方案(见第2部分)
  38. 阻塞缓冲区(见方法3)
  39. 抗体稀释剂(见配方4)

设备

  1. 平衡
  2. 加热台(,例如,MEDAX,型号:13801)
  3. 动物理发剪(例如,Aesculap Exacta Tierschermaschine,B.Braun Medical,Aesculap,目录号:GT415)
  4. 组织处理器(例如,Leica Biosystems,型号:Leica TP1020)
  5. 数码相机(如,Panasonic,型号:Lumix DMC-LX7)
  6. 荧光显微镜:配备AxioCam数码彩色相机和AxioVision软件v4.7(蔡司)的Zeiss Axiophot显微镜
  7. 蒸汽锅( 。,Braun MultiGourmet蒸锅,Braun,型号:FS 20)
  8. 幻灯片染色盘(例如,用于带有黑色盖的底部,Simport,目录号:M920-2的20张载玻片的StainTray TM系统)

软件

  1. ImageJ
  2. Graphpad棱镜
  3. AxioVision(蔡司)

程序

  1. Arthus反应在背部皮肤
    1. 称重实验性C57BL / 6J小鼠,根据体重为5μl/ g体重(例如,150μg,对于30g小鼠),腹膜内注射麻醉溶液(参见配方1)。预计小鼠麻醉至少30分钟。在麻醉期间将小鼠放置在加热台(39℃)上以保持体温。
    2. 用动物理发器刮背背皮肤上的头发。
    3. 可选:应用治疗剂和相应的对照。
      注意:
      1. 治疗剂的施用方法和持续时间需要针对个体治疗条件来确定。例如,为了研究间充质干细胞(MSC)对IC介导的血管炎的作用,将每次注射的100μlPBS中的2.5×10 5个脂肪组织来源的MSC皮内注射到剃掉老鼠的背部皮肤。
      2. 小鼠皮内注射100μlPBS作为对照。将这种体积注射到小鼠耳朵中是不可能的。
      3. 注射MSCs半小时后可诱导Arthus反应。
    4. 静脉注射(或腹膜内注射)100μl埃文斯蓝-BSA溶液(见配方2)。
      注:与i.v相比注射,i.p.注射导致抗BSA抗体的注射部位皮肤中的伊文思蓝区域更小,但是抗BSA和兔血清对照之间的差异是明显的(图S1 )。因此,埃文斯蓝-BSA溶液的注射也可以用于此程序。
    5. 皮内注射40μl兔抗BSA抗体(未稀释的全抗血清,浓度在不同批次,在我们的实验中从3-5mg / ml变化)到感兴趣的背侧皮肤(例如已经治疗过测试治疗剂的区域,如用AT-MSC或相应的对照)。在对照小鼠中,皮内注射40μl兔血清。
    6. 四小时后,量化伊文思蓝血管渗漏的程度,牺牲小鼠,收获皮肤标本(视频1),并立即拍照(图1B)。

      视频1
    7. 分析皮肤中的伊文思蓝区域,指示由于BSA /抗BSA复合物形成导致的血管损伤程度,ImageJ(详见数据分析)(图1)。


      图1.小鼠背部皮肤中IC介导的血管炎的诱导和数据分析A.数据分析的流程图; B.将100μlPBS或2.5×10 5脂肪组织衍生的间充质干细胞(MSC)皮内注射到小鼠的剃背皮的两侧。 Arthus反应是由i.v引起的。注射含有1%伊文思蓝和2%BSA的100μlPBS溶液。之后,将40μl抗-BSA抗体皮内注射到注射MSC或PBS的区域。 4小时后,处死小鼠,收集皮肤标本并数字化拍照。 C.使用ImageJ,RGB格式的原始照片被转换为HSB格式的插件“颜色空间转换器”。 D.分解通道后,保留绿色通道进行伊文思蓝色区域分析。 d”。或者,将RGB照片转换为插件“RGB到CYMK”的CYMK格式,保留青色通道进行分析。 E.二元图像是用黑色的伊文思蓝色区域创建的。 F和G.设置测量和粒子分析。 H.粒子分析显示皮肤中的伊文思蓝区域。 I.结果由GraphPad Prism作为散点图呈现。与PBS对照相比,皮内注射MSC导致伊文思蓝染色的面积显着减少了52.1%(15.8对比33.0AU)。 **, 0.01通过Mann-Whitney测试; AU,任意单位。

  2. 嗜中性粒细胞的组织学和免疫荧光
    注意:H&amp; E染色和嗜中性粒细胞激活标记的免疫荧光染色的组织学检查是可选的,但是为了确认所观察到的组织损伤确实是由于嗜中性粒细胞募集和激活所致,所以推荐使用。&lt; emr />
    1. 没有埃文斯蓝色的Arthus反应
      遵循程序A.在步骤A4中,将Evans blue-BSA溶液替换为在PBS中的无菌过滤的2%BSA。在步骤A6中,将收获的皮肤标本在4℃的10%福尔马林溶液中固定过夜。用组织处理器处理固定的组织,用于脱水和清除并包埋在石蜡块中,并用切片机切成5μm切片(使福尔马林固定石蜡包埋的[FFPE]切片的细节可以参考现有文献例如,Canene-Adams,2013)。
    2. H&amp; E染色(图2)
      1. 将石蜡切片在50-60°C加热30分钟。

      2. 在二甲苯中放置10分钟2次
      3. 在100%,95%,80%,70%乙醇中水解3分钟,每次2次。
      4. 在蒸馏水中孵育5分钟。
      5. 在Mayer的苏木素中孵育10分钟。

      6. 在冷的自来水中清洗5分钟
      7. 用蒸馏水冲洗(将载玻片在蒸馏水中浸几次)。
      8. 在曙红Y(+ 1滴醋酸/ 100毫升)中孵育2分钟。
      9. 用蒸馏水冲洗。

      10. 在80%,95%,100%乙醇中分别脱水5分钟
      11. 用Roti-histol清除。
      12. 用Roti-Histokitt装载。


        图2.诱导IC介导的血管炎后FFPE皮肤切片的H&amp; E染色小鼠皮内注射PBS或MSC,并通过静脉注射进行Arthus反应。注射含有2%BSA的100μlPBS溶液。之后,将抗BSA抗体皮内注射到注射了MSC或PBS的区域。小鼠接受兔血清作为健康对照。 4小时后,处死小鼠,收集皮肤标本并加工成5μm石蜡包埋切片。显示了每组4只小鼠的皮肤切片的H&amp; E染色的代表性图片。白色箭头表示血管。比例尺= 50微米。用PBS注射Arthus反应的小鼠的真皮显示红血球和嗜中性粒细胞包围的血管的严重损伤(B)。 MSC的预处理减少了血管破坏(C),并且组织学类似于来自健康对照小鼠(A)的皮肤样本。

    3. 嗜中性粒细胞标志物(Ly6G),嗜中性粒细胞胞外陷阱(NETs),嗜中性粒细胞弹性蛋白酶(NE)和髓过氧化物酶(MPO)的免疫荧光染色。
      1. 按照步骤B2a-B2d。
      2. 在步骤B3a中,制备抗原修复缓冲液:20ml 10x目标修复液+ 180ml蒸馏水,并在蒸汽锅中加热缓冲液。
      3. 抗原提取:在蒸汽锅中加热缓冲液15分钟(在塑料室中)。
      4. 冷却10分钟,放入PBS中5分钟。
      5. 渗透性:将切片置于0.25%Triton X-100中10分钟(0.5ml Triton X-100在200ml PBS中)。
      6. 每5分钟用PBS清洗3次。

      7. 在封闭缓冲液(参见配方3)中室温孵育1小时
      8. (图3A-3C)或抗NE(1:100稀释在抗体稀释剂中[参见配方4])在4°C孵育过夜, 200稀释在抗体稀释剂中)(图3D-3F)。
      9. 用PBS清洗3次,每次5分钟。
      10. (图3A-3C)或AF488结合的驴抗大鼠IgG(在抗体稀释剂中1:200稀释)(图3A-3C),在室温下用第一种二抗孵育细胞1小时,兔IgG(在抗体稀释剂中1:200稀释)(图3D-3F)。
      11. PBS洗3次,每次5分钟。避免直接光线照射。
      12. (图3A-3C)或抗MPO(1μg/ ml)的抗DNA /组蛋白H1抗体(在抗体稀释剂中1:100稀释)(图3A-3C),在室温下孵育第二个第一抗体的切片2小时:在抗体稀释液中稀释50倍)(图3D-3F)。
      13. 用PBS清洗3次,每次5分钟。
      14. (图3A-3C)或AF555结合的驴抗小鼠IgG(在抗体稀释剂中1:200稀释)(图3A-3C),在室温下用第二个二抗孵育细胞1小时,山羊IgG(在抗体稀释液中1:200稀释)(图3D-3F)。
      15. PBS洗3次,每次5分钟。避免直接光线照射。

      16. 在室温下用DAPI溶液(1:5,000稀释于PBS中)孵育3分钟。
      17. 用PBS冲洗载玻片,风干。避免直接的光照。
      18. 用荧光安装介质安装盖玻片。幻灯片可以保存在4°C。
      19. 荧光显微镜。


        图3.诱导IC介导的血管炎后FFPE皮肤切片上的嗜中性粒细胞活化标记的免疫荧光染色小鼠皮内注射PBS或MSC,并通过静脉注射进行Arthus反应。注射含有2%BSA的100μlPBS溶液。之后,将抗BSA抗体皮内注射到注射了MSC或PBS的区域。小鼠接受兔IgG作为健康对照。 4小时后,处死小鼠,收集皮肤标本并加工成5μm石蜡包埋的切片。 (绿色)和NET标记DNA /组蛋白-1(红色)(AC)的免疫染色以及NE(绿色)和MPO(红色)(DF)(DF)的免疫染色的每组4只小鼠的皮肤切片的代表性照片)被显示。在免疫染色中核用DAPI(蓝色)复染。比例尺= 50微米。用PBS注射Arthus反应的小鼠的真皮表现出与NET(B),NE和MPO在组织(E)中的嗜中性粒细胞的大量浸润,这非常类似于人血管炎的特征。相比之下,皮内注射MSCs显着降低真皮(F)中的NETs(C)和NE + MPO +中性粒细胞的数量,这与皮肤样本相似健康对照小鼠(A和D)。

数据分析

用ImageJ分析Arthus反应皮肤中伊文思蓝的面积。分析流程图如图1A所示。

  1. 下载ImageJ插件“颜色空间转换器”下的“颜色”类别:
    https://imagej.nih.gov/ij/plugins/color-space -converter.html 。将“Color_Space_Converter.jar”保存在插件文件夹或子文件夹中,例如,工具:... \ ImageJ \ plugins \ Tools
  2. 打开原始的RGB照片。 '插件 - 工具 - 色彩空间转换器':选择RGB到HSB,保留其他设置,单击'确定'(图1C)。
  3. “图像颜色分割通道”。使用绿色通道进行区域分析(图1D),关闭另外两个通道(红色和蓝色)。
    注:备选步骤1-3;替代插件被使用:“RGB到CYMK”。
    https://imagej.nih.gov/ij/plugins下载插件/cmyk/index.html 。通道拆分后,保持青色(C)通道(图1D')转换为二进制,关闭其他通道(M,Y,K)。
  4. “图像 - 调节门限”。检查“黑暗的背景”,并将所有照片的阈值设置为固定值并应用。这将创建一个二进制(黑白)图像,黑色的伊文思蓝色区域(图1E)。设置背景值以在伊文思蓝信号的充足强度上达到平衡,但是使背景最小。
  5. 在二进制中,“分析 - 设置测量”:选中“区域”,单独保留其他选项,点击“确定”(图1F)。
  6. “分析 - 分析粒子”:设置“大小(像素^ 2)”:例如,2000-infinity。 “显示”:选择“覆盖”。
    检查“显示结果”(图1G和1H)(尺寸值可能需要调整更小的区域,取决于最后计算的内容,详情请参阅注释部分)。您可能需要手动删除非伊文思蓝色背景区域的数据。
  7. 将结果保存在GraphPad Prism中进行分析。建议使用每个治疗组至少5只小鼠进行该测定。数据可以通过GraphPad Prism作为散点图(垂直)呈现。曼 - 惠特尼检验可以用于显着性检验(图1I)。

笔记

  1. ImageJ数据分析故障排除
    1. 如果您没有看到打开结果的窗口,则忘记在“分析 - 分析粒子”中检查“显示结果”。
    2. 如果您没有看到二进制文件中哪个区域对应哪个区域,则忘记在“分析 - 分析粒子”中将“显示”设置为“覆盖”。
    3. 如果有数百个非常小的区域,应该在“分析 - 分析粒子”中增加粒子的最小尺寸。
    4. 如果其中一个黑色区域未被分析,则应在“分析 - 分析粒子”中增加粒子的最小尺寸。

食谱

  1. 麻醉解决方案(基于氯胺酮和甲苯噻嗪)
    每10ml麻醉溶液中含有:
    4毫升Ketanest S 25毫克/毫升
    0.5毫升2%rompun
    5.5毫升无菌PB
  2. 伊文思蓝BSA溶液(可以保持在4°C一个星期)
    1%伊文思蓝+ 2%BSA在PBS中,通过一个0.22微米的过滤器
  3. 阻塞缓冲区(准备新鲜)
    PBS中含有5%BSA + 5%山羊血清的PBS
  4. 抗体稀释剂(准备新鲜)
    含1%BSA的PBS

致谢

这项工作得到了巴登 - 符腾堡州基金会(P-BWS-ASII / 15),欧盟委员会(CASCADE HEALTH-FP7-223236)和德国研究基金会(SFB1149)对KS-K研究基金的资助。乌尔姆大学医学院的Baustein计划(LSBN.0100)到DJ部分数据是根据Jiang等人的研究调整和修改的,2016年。作者指出没有潜在的利益冲突。

参考

  1. Canene-Adams,K.(2013)。 福尔马林固定石蜡包埋组织的免疫组织化学方法方法Enzymol 533:225-233。
  2. Dallegri,F.,Patrone,F.,Frumento,G.,Ballestrero,A.和Sacchetti,C。(1984)。 固定化聚集IgG引发的中性粒细胞介导的细胞毒性:体外 免疫复合物疾病期间的细胞损伤模型 J Clin Immunol 4(6):439-444。
  3. Feld,M.,Goerge,T.,Hillgruber,C.,Steingraber,A.K.,Fastrich,M.,Shpacovitch,V。和Steinhoff,M。(2012)。 α-1-抗胰蛋白酶和IFN-γ通过调节白细胞降低IC介导的血管炎的严重程度招聘。。 J Invest Dermatol 132(9):2286-2295。
  4. Goerge,T.,Ho-Tin-Noe,B.,Carbo,C.,Benarafa,C.,Remold-O'Donnell,E.,Zhao,B.Q.,Cifuni,S.M。和Wagner,D.D。(2008)。 炎症导致血小板减少症出血血液111(10 ):4958-4964。
  5. Incani,R.N.和McLaren,D.J。(1981)。 嗜中性粒细胞介导的曼氏血吸虫鞘膜虫体外细胞毒性研究补体和/或抗体依赖性粘附和杀伤的动力学。 寄生虫免疫 3(2):107-126。
  6. Jiang,D.,Muschhammer,J.,Qi,Y.,Kugler,A.,de Vries,JC,Saffarzadeh,M.,Sindrilaru,A.,Beken,SV,Wlaschek,M.,Kluth,MA,Ganss, C.,Frank,NY,Frank,MH,Preissner,KT和Scharffetter-Kochanek,K.(2016)。 抑制嗜中性粒细胞介导的组织损伤 - 一种间充质干细胞的新技能 干细胞 34(9):2393-2406。
  7. Saffarzadeh,M.,Juenemann,C.,Queisser,M.A.,Lochnit,G.,Barreto,G.,Galuska,S.P.,Lohmeyer,J。和Preissner,K.T。(2012)。 嗜中性粒细胞胞外陷阱直接诱导上皮和内皮细胞死亡:组蛋白的主要作用。 PLoS One 7(2):e32366。
  8. Sindrilaru,A.,Seeliger,S.,Ehrchen,J.M.,Peters,T.,Roth,J.,Scharffetter-Kochanek,K.and Sunderkotter,C.H。(2007)。 免疫复合物介导的血管炎小鼠模型中血管损伤和CD18相关性的位点< / J> Invest Dermatol 127(2):447-454。
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引用:Jiang, D., de Vries, J. C., Muschhammer, J., Sindrilaru, A. and Scharffetter-Kochanek, K. (2017). Mouse Model of Immune Complex-mediated Vasculitis in Dorsal Skin and Assessment of the Neutrophil-mediated Tissue Damage. Bio-protocol 7(24): e2660. DOI: 10.21769/BioProtoc.2660.
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