Chapter 30 Detection of Soluble CR3 (CD11b/CD18) by Time-Resolved Immunofluorometry Gitte Krogh Nielsen and Thomas Vorup-Jensen Abstract In the cell membrane complement receptor 3 (CR3) consists of one alpha chain (CD11b) and one beta chain (CD18). CR3 participates in many immunological processes, especially those involving cell migration, adhesion, and phagocytosis of complement-opsonized microbes. Recent findings of soluble CR3 in body fluids and in culture supernatant from experiments in vitro point to the involvement of ecto domain shedding as a part of the CR3 biology. To monitor such shedding on a quantitative basis, we have developed time-resolved immunofluorometric assays (TRIFMA) to detect soluble CD11b and CD18 in plasma or serum of either human or murine origin. Compared with most enzyme-linked immunosorbent assays methodologies, TRIFMA possesses prominent advantages, including better dynamic range and reproducibility. These assays may contribute to the understanding of the role of shedding of CR3 and other cell adhesion molecules in human disease and animal models involving inflammation. Key words Complement, Integrin, CR3, CD11b, CD18, TRIFMA
1
Introduction The complement receptor 3 (CR3) is a member of the β2 subfamily of integrins consisting of heterodimeric cell membrane-anchored receptors. The β2 subfamily comprises four known members that share a common β2 subunit associated non-covalently with either of the four distinct, but structurally similar, α-subunits to form the receptors CR3 (also named αMβ2, CD11b/CD18, or Mac-1), αLβ2 (also known as LFA-1 or CD11a/CD18), αXβ2 (CD11c/CD18, CR4, p150,95), and αDβ2 (CD11d/CD18) [1]. The β2 integrins play a crucial role in the immune system as demonstrated in patients with leukocyte adhesion deficiency (LAD). Such individuals have a global deficiency in phagocyte-mediated acute inflammatory responses due to deficiency of CD18 [1–3]. Consequently, they suffer from recurrent infections due to the malfunction of their macrophages and neutrophil granulocytes [4, 5].
Mihaela Gadjeva (ed.), The Complement System: Methods and Protocols, Methods in Molecular Biology, vol. 1100, DOI 10.1007/978-1-62703-724-2_30, © Springer Science+Business Media New York 2014
355
356
Gitte Krogh Nielsen and Thomas Vorup-Jensen
The CR3 is an abundant β2 integrin receptor in the cell membrane of neutrophiles, macrophages, and monocytes and participates in many immunological processes, especially those involving cell migration, adherence, and phagocytosis [1, 6–8]. Via the I-domain located in the N-terminal ectodomain of the α-subunit, CR3 binds a wide variety of ligands in a divalent cationdependent manner [9–11]. Notably, CR3 binds the fragment of complement component C3 generated by complement factor I cleavage, namely, iC3b [12, 13], providing one of the links between the complement system and cellular immunity. By recognizing iC3b-opsonized particles the CR3 triggers phagocytosis resulting in clearance of pathogens and apoptotic cells [14, 15]. Other ligands include the intercellular adhesion molecule (ICAM) family of intercellular adhesion molecules [11]. The binding of CR3 to ICAMs plays a key role in the molecular mechanisms supporting leukocyte migration and diapedesis through the endothelial barrier and accumulation at sites of tissue injury [16, 17]. The list of CR3 ligands has gradually been expanding, however, and now includes more than 50 molecules of considerable structural and chemical diversity [18, 19]. The ability of the receptor to interact with proteins, polymers of nucleic acids, and lipopolysaccharides was suggested to originate from a relatively high affinity for at least certain acidic moieties such as the γ-COOH of the glutamate side chains as well as the carboxyl groups found in the uronic acid residues in heparin [20, 21]. In the past, the main focus has been on the cell surface expression of CR3 and ligand binding properties in connection to its role in leukocyte adhesion and migration. However, recently the finding of soluble (s)CR3 in various body fluids suggested the functional importance of shedding. Results from our laboratory demonstrated that the concentration of sCD11/CD18 is elevated in synovial fluid as consequence inflammatory arthritis while such fluid under conditions with less or no inflammation has a significantly lower concentration sCD11/CD18 [22]. Systemic alterations, i.e., in the plasma concentration, were also found in inflamed patients compared with healthy volunteers. Although the distribution of the four alpha chains in these complexes is unclear, CD11a was easily detectable in complexes found in human plasma while, by contrast, CD11b was barely detectable and CD11c was not detected. However, in addition to the relative concentration of these species, this finding may at least in part reflect properties of the monoclonal antibodies utilized for detection as well. Other investigations presented evidence that proteolytic cleavage of the extracellular domain serves as an important mechanism for leukocyte detachment after initial cell adhesion and transmigration in both humans and mice [23]. Kinetic studies showed that the cleavage of CD11b is positively correlated with polymorphonuclear leukocyte detachment and subsequent transmigration [24–27].
Detection of Soluble CR3 (CD11b/CD18) by Time-Resolved Immunofluorometry
357
Cross-immunoprecipitation using CD11b and CD18 antibodies further suggested that the cleaved CD11b and CD18 fragments were at least partially associated with one another, suggesting a potential biological function for the soluble integrin fragments [23]. Specific cleavage of the CD18 ectodomain by MMP-9 further supports a functional importance of soluble β2 integrins [28]. Likewise, specific anti-CD11b antibodies that recognize the I-domain are applicable for detecting the soluble ectodomain suggesting that these possess a ligand binding activity [23]. Indeed, in humans sCD11/CD18 was found to be able to bind ICAM-1 immobilized on plastic surfaces, although in this case the binding was most likely contributed by sCD11a/CD18 [22]. In light of the above-described findings and the possible functional importance of CR3 ectodomain shedding in different conditions, we have developed a time-resolved immunofluorometric assay (TRIFMA) to detect sCD11b and sCD18 in serum of both human and murine origin (Fig. 1). The TRIFMA assay is a method for detection of specific analytes very similar to ELISA but with prominent advantages. The long-lasting fluorescence of the europium ions is measured by photon counting with better sensitivity, dynamic range, and reproducibility as compared to the enzymatic based reaction of ELISAs. In addition, the fluorescence emitted from the europium ions upon reading can be measured following storage of the microtiter plates at 4 °C for several days. We believe that these assays may add new tools for understanding the role of cell adhesion molecules in human disease and animal models involving inflammation, notably those involving CR3.
2
Materials All buffers and solutions are made with analytic grade reagents and ultrapure water prepared by purifying deionized water to attain a resistivity of 18 MΩ cm at 25 °C. Buffers are typically supplemented with 10 mM NaN3 to preserve sterility and are stored at 4 °C until use. 1. 96-Well microtiter plates, e.g., FluoroNunc Maxisorp™ microtiter plates (see Note 1). 2. Plastic container. 3. Paper towels. 4. Coating antibody: Monoclonal (m) antibody (Ab) anti-CD11b (e.g., mAb anti-mouse CD11b, clone 5c6) or anti-CD18 antibodies of choice (mAbs, anti-human CD18 antibody, clone KIM185, and anti-mouse CD18 antibody, clone 313903) (see Table 1). 5. Detection antibody: Biotin-labeled mAb anti-CD11b-antibody (e.g., mAb anti-mouse CD11b antibody, clone M1/70.15) or
Gitte Krogh Nielsen and Thomas Vorup-Jensen
a
600
400
200
0
100
murine sCD18 human sCD18
80 60 40 20
50
25
.5 12
5
25 6.
3
12 3.
1
56 1.
1
0.
78
5
39
09 0.
0.
8
0
0.
b
murine sCD11b
19
sCD11b (counts per sec x 10-3)
800
sCD18 (counts per sec x 10-3)
358
Serum conc. (%, v/v)
Fig. 1 Detection of sCD11b and sCD18 by TRIFMA. Serum samples from either male C57BL/6 mice or human volunteers were titrated as indicated in the lower abscissa axis (in serum volume percentage). (a) Serum samples from male C57BL7/6 mice were applied to wells coated with rat mAb anti-mouse CD11b antibody (clone 5c6) followed by development with biotinylated mAb anti-mouse CD11b antibody (clone M1/70.15). (b) Serum samples from human volunteers were applied to wells coated with KIM185 and developed with biotinylated antibody KIM127. Rat mAb anti-mouse CD18 antibody (clone 313903) was used to coat wells to detect CD18 in serum from male C57BL/6 mice, whereas the biotinylated rat mAb anti-mouse CD18 antibody (clone C71/16) was used for development
biotin-labeled monoclonal anti-CD18-antibody of choice (e.g., mAb anti-human CD18 antibody, clone KIM127, and anti-mouse CD18 antibody, clone C71/16) (see Table 1). 6. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.5 mM KH2PO4, 6.9 mM K2HPO4⋅3H2O, pH 7.4. 7. Tris-buffered saline (TBS): 10 mM Tris–HCl, pH 7.4, 145 mM NaCl, 15 mM NaN3.
Thioglycollate-elicited peritoneal macrophages (TPM)
T cell-enriched splenocytes from B10 mice
Monoclonal rat antimouse CD11b (5C6)
Monoclonal rat antimouse CD11b (M1/70.15)
Anti-mouse CD11b
Monoclonal rat anti-mouse Cell membrane glycoproteins from CD18 (C71/16) mouse T-cell lymphoma BW5147
Monoclonal rat antimouse CD18 (313903)
CHO-derived rmIntegrin β2 (Gln24-Asn702) accession # P11835
Human CR3 immunopurified from human white cell lysate. Human CR3 immunized BALB/c mouse spleen cells fused with Sp2/0 cells
Monoclonal mouse anti-human CD18 (KIM127)
Anti-mouse CD18
Human CR3 immunopurified from human white cell lysate. Human CR3 immunized BALB/c mouse spleen cells fused with Sp2/0 cells
Immunogen
Monoclonal mouse anti-human CD18 (KIM185)
Anti-human CD18
Antibody (clone)
IgG2b
Recognizes the murine CD11b cell surface antigen
Recognizes mouse complement type 3 receptor (CR3) and precipitates a heterodimer of 165 and 95 kD
CD18
IgG2a, κ
IgG2b
Integrin beta 2/CD18
Integrin β2/CD18
IgG1, κ
IgG2a
Integrin β2/CD18
Specificity
IgG1, κ
Isotype (Ig class)
Table 1 List of recommended anti-CD11band anti-CD18 monoclonal Abs and their characteristics
Human, mouse, rabbit
Human, mouse
Mouse
Mouse
Human
Human
Reactivity
[32, 33]
[32]
[30, 31]
[29]
[22]
[22]
References
AbD Serotec (Cat# MCA74)
AbD Serotec (Cat# MCA711)
BD Pharmingen (557439)
R&D Systems (MAB2618)
Provider (cat. no.)
359
360
Gitte Krogh Nielsen and Thomas Vorup-Jensen
8. TBS/Tw: TBS with 0.05 % (v/v) Tween-20. 9. Human serum albumin (HSA) (see Note 2). 10. Serum or plasma samples for analysis (store in aliquots at −80 °C until use). 11. Heat-aggregated human IgG (see Note 3). 12. Bovine IgG, rat IgG, and mouse IgG. 13. Assay binding buffer: TBS/tw, 1 mM CaCl2, 1 mM MgCl2, 100 μg/ml heat-aggregated human IgG, 100 μg/ml bovine IgG, 100 μg/ml rat IgG, 100 μg/ml mouse IgG (see Note 4). 14. Biotinylated Ab binding buffer: TBS/Tw, 1 mM CaCl2, 1 mM MgCl2. 15. Streptavidin-Eu3+. 16. TBS/Tw/EDTA: TBS with 0.05 % (v/v) Tween 20 and 25 μM EDTA. 17. Enhancement solution: Mix 5.7 ml of glacial acetic acid with a sufficient amount of 0.1 M potassium hydrogen phthalate to reach pH 3.2. Add 1 ml Triton X-100, and adjust the volume to 1 l with distilled water. Just before use, add 2 μl of a solution containing 15 μM 2-naphthoyltrifluoracetate (2-NTA) and 50 μM tri-o-octylphosphine oxide (TOPO) per ml of the acetic acid/potassium hydrogen phthalate mixture (see Note 5).
3
Methods Carry out all procedures at room temperature unless otherwise specified.
3.1 Detection of Soluble CR3 by TRIFMA
Detection of sCD11b and sCD18 in serum samples is carried out by TRIFMA using a “sandwich” technique with pairs of polystyrenecoated and biotinylated mAbs applied for the detection of either the CD11b chain or the CD18 chain of CR3. 1. Coat the wells of a 96-well microtiter plate by adding the captured mAb diluted in PBS buffer to each well using a total volume of 100 μl per well. The mAb anti-mouse CD11b (clone 5c6) is used in a concentration of 0.5 μg per ml PBS, whereas the optimal concentration of mAb anti-mouse CD18 (clone 313903) is 1 μg per ml PBS. In assay for human samples the mAb anti-human CD18 antibody (clone KIM185) is used at a concentration of 1 μg per ml PBS (see Table 1 for information about the antibodies). 2. Incubate at 4 °C overnight in a moist chamber (see Note 6). 3. Empty the wells by tapping the plate on a layer of paper towels, and block nonspecific binding by adding 200 μl of TBS containing 0.05 % (v/v) Tween 20 to each well. Incubate for 1 h at
Detection of Soluble CR3 (CD11b/CD18) by Time-Resolved Immunofluorometry
361
20 °C. After incubation, empty wells and wash three times with 200 μl of TBS containing 0.05 % (v/v) Tween 20 (see Note 7). 4. Dilute samples of serum (or plasma) 1:10 or 1:20 in binding buffer for antigen assay. Add 100 μl to each well of the coated plate. Incubate in moist chamber overnight at 4 °C. At the end of incubation, empty wells by repeated tapping of the plate on a layer of paper towels, and then wash wells three times with TBS containing 0.05 % (v/v) Tween 20. 5. Dilute the biotinylated mAb in TBS containing 0.05 % (v/v) Tween 20. Rat anti-CD11b antibody (clone M1/70.15) is diluted to 0.25 μg/ml, whereas rat anti-CD18 antibody (clone C71/16) is used at a concentration of 1 μg/ml. The biotinylated mAb anti-human antibody (KIM127) should be used in a concentration of 1 μg/ml. Add 100 μl of the diluted antibody to each well and incubate for 1–2 h at 20 °C in the moist chamber. At the end of incubation, empty wells by tapping the plate repeatedly on a layer of paper towels, and then wash wells three times with TBS containing 0.05 % (v/v) Tween 20. 6. Dilute streptavidin-Eu3+ appropriately (e.g., 1:1,000) in TBS containing 0.05 % (v/v) Tween 20 and 25 μM EDTA. Add 100 μl to each well and incubate for 1–2 h at 20 °C in the moist chamber. At the end of incubation, empty wells by repeated tapping of the plate on a layer of paper towels, and then wash wells three times with TBS containing 0.05 % (v/v) Tween 20. 7. Add 200 μl enhancement solution per well, and incubate plate for 5 min with shaking at room temperature. Read the signals developed in a machine equipped for TRIFMA measurements (see Note 8).
4
Notes 1. For example, FluoroNunc Maxisorp™ microtiter plates with a low background fluorescence. The maxisorp surface is a modified, highly charged polystyrene surface with high affinity to molecules with polar or hydrophilic groups, which implies that when coating with antibodies the hydrophilic Fc region of the antibody is more likely to be bound, thereby leaving the fab region intact for detection of a specific analyte. 2. HSA can be added to the TBS/TW buffer used as blocking buffer to reduce background fluorescence signal. When necessary, we use HSA at a concentration of 1 mg/ml to reduce background fluorescence noise signal. 3. The IgG is heat-aggregated prior to use in the assay by incubation of TBS with 10 mg/ml IgG at 63 °C for 30 min followed
362
Gitte Krogh Nielsen and Thomas Vorup-Jensen
by centrifugation for 10 min at 3,000 × g to remove insoluble complexes (grossly aggregated IgG). The supernatant is then used. Addition of heat-aggregated IgG reduces interference by rheumatoid factor (RF) in the assay. 4. Since immunoglobulin-reactive antibodies are found in sera from humans as well as other species, the inclusion of aggregated human IgG and IgGs from mouse, rat, and cow is a mean of avoiding an influence of the immunoglobulins binding to antibodies applied for antigen detection. The presence of these immunoglobulin-reactive antibodies may cause a false-positive signal through the binding and capture of the antibodies applied for antigen detection. We find that it is best to prepare the assay binding buffer fresh each time. 5. Store both the acetic acid/potassium hydrogen phthalate solution and the NTA/TOPO solutions up to 3 months at 4 °C. Once the solutions are mixed, the complete enhancement solution should be used within a month. Furthermore, it is advisable to check the background fluorescence signal of the enhancement buffer just after preparation. Counts per second should not exceed 1,000. 6. The humid chamber consists of a closed plastic container with a moist paper towel at the bottom. 7. If the plates are to be stored for later use, add 200 μl TBS per well and store at 4 °C in a moist chamber. 8. Measurements of TRIFMA signals can be done using a Victor® plate reader available from PerkinElmer.
Acknowledgements We would like to thank Drs. Jens C. Jensenius and Steffen Thiel, Dept. of Biomedicine, Aarhus University, for their support and excellent advice on applications of TRIFMA. This work was financially supported by grants from the Lundbeck Foundation (LUNA), the Danish Multiple Sclerosis Association, and the Danish Council for Independent Research | Medical Sciences. References 1. Arnaout MA (1990) Structure and function of the leukocyte adhesion molecules CD11/ CD18. Blood 75(5):1037–1050 2. Hibbs ML, Wardlaw AJ, Stacker SA, Anderson DC, Lee A, Roberts TM, Springer TA (1990) Transfection of cells from patients with leukocyte adhesion deficiency with an integrin beta subunit (CD18) restores lymphocyte functionassociated antigen-1 expression and function. J Clin Invest 85(3):674–681
3. Wardlaw AJ, Hibbs ML, Stacker SA, Springer TA (1990) Distinct mutations in two patients with leukocyte adhesion deficiency and their functional correlates. J Exp Med 172(1):335–345 4. Kishimoto TK, Hollander N, Roberts TM, Anderson DC, Springer TA (1987) Heterogeneous mutations in the beta subunit common to the LFA-1, Mac-1, and p150,95 glycoproteins cause leukocyte adhesion deficiency. Cell 50(2):193–202
Detection of Soluble CR3 (CD11b/CD18) by Time-Resolved Immunofluorometry 5. Anderson DC, Springer TA (1987) Leukocyte adhesion deficiency: an inherited defect in the Mac-1, LFA-1, and p150,95 glycoproteins. Annu Rev Med 38:175–194 6. Dana N, Styrt B, Griffin JD, Todd RF 3rd, Klempner MS, Arnaout MA (1986) Two functional domains in the phagocyte membrane glycoprotein Mo1 identified with monoclonal antibodies. J Immunol 137(10):3259–3263 7. Ross GD, Yount WJ, Walport MJ, Winfield JB, Parker CJ, Fuller CR, Taylor RP, Myones BL, Lachmann PJ (1985) Disease-associated loss of erythrocyte complement receptors (CR1, C3b receptors) in patients with systemic lupus erythematosus and other diseases involving autoantibodies and/or complement activation. J Immunol 135(3):2005–2014 8. Park JY, Arnaout MA, Gupta V (2007) A simple, no-wash cell adhesion-based high-throughput assay for the discovery of small-molecule regulators of the integrin CD11b/CD18. J Biomol Screen 12(3):406–417 9. Lee JO, Rieu P, Arnaout MA, Liddington R (1995) Crystal structure of the A domain from the alpha subunit of integrin CR3 (CD11b/ CD18). Cell 80(4):631–638 10. Beller DI, Springer TA, Schreiber RD (1982) Anti-Mac-1 selectively inhibits the mouse and human type three complement receptor. J Exp Med 156(4):1000–1009 11. Diamond MS, Staunton DE, de Fougerolles AR, Stacker SA, Garcia-Aguilar J, Hibbs ML, Springer TA (1990) ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18). J Cell Biol 111(6 Pt 2):3129–3139 12. Rosen H, Law SK (1990) The leukocyte cell surface receptor(s) for the iC3b product of complement. Curr Top Microbiol Immunol 153:99–122 13. Ross GD, Vetvicka V (1993) CR3 (CD11b, CD18): a phagocyte and NK cell membrane receptor with multiple ligand specificities and functions. Clin Exp Immunol 92(2):181–184 14. Tsuji S, Kaji K, Nagasawa S (1994) Activation of the alternative pathway of human complement by apoptotic human umbilical vein endothelial cells. J Biochem 116(4):794–800 15. Ezekowitz RA (2002) Local opsonization for apoptosis? Nat Immunol 3(6):510–512 16. Ross GD (2000) Regulation of the adhesion versus cytotoxic functions of the Mac-1/CR3/ alphaMbeta2-integrin glycoprotein. Crit Rev Immunol 20(3):197–222 17. Dunne JL, Collins RG, Beaud et al, Ballantyne CM, Ley K (2003) Mac-1, but not LFA-1, uses intercellular adhesion molecule-1 to mediate slow leukocyte rolling in TNF-alpha-induced inflammation. J Immunol 171(11):6105–6111 18. Vorup-Jensen T (2012) On the roles of polyvalent binding in immune recognition: perspectives in the nanoscience of immunology and
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
363
the immune response to nanomedicines. Adv Drug Deliv Rev 64(15):1759–1781 Tan SM (2012) The leucocyte beta2 (CD18) integrins: the structure, functional regulation and signalling properties. Biosci Rep 32(3): 241–269 Vorup-Jensen T, Carman CV, Shimaoka M, Schuck P, Svitel J, Springer TA (2005) Exposure of acidic residues as a danger signal for recognition of fibrinogen and other macromolecules by integrin alphaXbeta2. Proc Natl Acad Sci U S A 102(5):1614–1619 Vorup-Jensen T, Chi L, Gjelstrup LC, Jensen UB, Jewett CA, Xie C, Shimaoka M, Linhardt RJ, Springer TA (2007) Binding between the integrin alphaXbeta2 (CD11c/CD18) and heparin. J Biol Chem 282(42):30869–30877 Gjelstrup LC, Boesen T, Kragstrup TW, Jorgensen A, Klein NJ, Thiel S, Deleuran BW, Vorup-Jensen T (2010) Shedding of large functionally active CD11/CD18 Integrin complexes from leukocyte membranes during synovial inflammation distinguishes three types of arthritis through differential epitope exposure. J Immunol 185(7):4154–4168 Zen K, Guo YL, Li LM, Bian Z, Zhang CY, Liu Y (2011) Cleavage of the CD11b extracellular domain by the leukocyte serprocidins is critical for neutrophil detachment during chemotaxis. Blood 117(18):4885–4894 Evans BJ, McDowall A, Taylor PC, Hogg N, Haskard DO, Landis RC (2006) Shedding of lymphocyte function-associated antigen-1 (LFA-1) in a human inflammatory response. Blood 107(9):3593–3599 Mechtersheimer S, Gutwein P, Agmon-Levin N, Stoeck A, Oleszewski M, Riedle S, Postina R, Fahrenholz F, Fogel M, Lemmon V, Altevogt P (2001) Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins. J Cell Biol 155(4):661–673 Phillipson M, Heit B, Colarusso P, Liu L, Ballantyne CM, Kubes P (2006) Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade. J Exp Med 203(12): 2569–2575 Phillipson M, Heit B, Parsons SA, Petri B, Mullaly SC, Colarusso P, Gower RM, Neely G, Simon SI, Kubes P (2009) Vav1 is essential for mechanotactic crawling and migration of neutrophils out of the inflamed microvasculature. J Immunol 182(11):6870–6878 Vaisar T, Kassim SY, Gomez IG, Green PS, Hargarten S, Gough PJ, Parks WC, Wilson CL, Raines EW, Heinecke JW (2009) MMP-9 sheds the beta2 integrin subunit (CD18) from macrophages. Mol Cell Proteomics 8(5):1044–1060 Gomez IG, Tang J, Wilson CL, Yan W, Heinecke JW, Harlan JM, Raines EW (2012)
364
Gitte Krogh Nielsen and Thomas Vorup-Jensen
Metalloproteinase-mediated shedding of integrin beta2 promotes macrophage efflux from inflammatory sites. J Biol Chem 287(7):4581–4589 30. Springer TA (1990) Adhesion receptors of the immune system. Nature 346(6283):425–434 31. Springer TA (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76(2):301–314 32. Rosen H, Gordon S (1987) Monoclonal antibody to the murine type 3 complement
receptor inhibits adhesion of myelomonocytic cells in vitro and inflammatory cell recruitment in vivo. J Exp Med 166(6): 1685–1701 33. Halle A, Hornung V, Petzold GC, Stewart CR, Monks BG, Reinheckel T, Fitzgerald KA, Latz E, Moore KJ, Golenbock DT (2008) The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nat Immunol 9(8):857–865
1
Introduction The complement receptor 3 (CR3) is a member of the β2 subfamily of integrins consisting of heterodimeric cell membrane-anchored receptors. The β2 subfamily comprises four known members that share a common β2 subunit associated non-covalently with either of the four distinct, but structurally similar, α-subunits to form the receptors CR3 (also named αMβ2, CD11b/CD18, or Mac-1), αLβ2 (also known as LFA-1 or CD11a/CD18), αXβ2 (CD11c/CD18, CR4, p150,95), and αDβ2 (CD11d/CD18) [1]. The β2 integrins play a crucial role in the immune system as demonstrated in patients with leukocyte adhesion deficiency (LAD). Such individuals have a global deficiency in phagocyte-mediated acute inflammatory responses due to deficiency of CD18 [1–3]. Consequently, they suffer from recurrent infections due to the malfunction of their macrophages and neutrophil granulocytes [4, 5].
Mihaela Gadjeva (ed.), The Complement System: Methods and Protocols, Methods in Molecular Biology, vol. 1100, DOI 10.1007/978-1-62703-724-2_30, © Springer Science+Business Media New York 2014
355
356
Gitte Krogh Nielsen and Thomas Vorup-Jensen
The CR3 is an abundant β2 integrin receptor in the cell membrane of neutrophiles, macrophages, and monocytes and participates in many immunological processes, especially those involving cell migration, adherence, and phagocytosis [1, 6–8]. Via the I-domain located in the N-terminal ectodomain of the α-subunit, CR3 binds a wide variety of ligands in a divalent cationdependent manner [9–11]. Notably, CR3 binds the fragment of complement component C3 generated by complement factor I cleavage, namely, iC3b [12, 13], providing one of the links between the complement system and cellular immunity. By recognizing iC3b-opsonized particles the CR3 triggers phagocytosis resulting in clearance of pathogens and apoptotic cells [14, 15]. Other ligands include the intercellular adhesion molecule (ICAM) family of intercellular adhesion molecules [11]. The binding of CR3 to ICAMs plays a key role in the molecular mechanisms supporting leukocyte migration and diapedesis through the endothelial barrier and accumulation at sites of tissue injury [16, 17]. The list of CR3 ligands has gradually been expanding, however, and now includes more than 50 molecules of considerable structural and chemical diversity [18, 19]. The ability of the receptor to interact with proteins, polymers of nucleic acids, and lipopolysaccharides was suggested to originate from a relatively high affinity for at least certain acidic moieties such as the γ-COOH of the glutamate side chains as well as the carboxyl groups found in the uronic acid residues in heparin [20, 21]. In the past, the main focus has been on the cell surface expression of CR3 and ligand binding properties in connection to its role in leukocyte adhesion and migration. However, recently the finding of soluble (s)CR3 in various body fluids suggested the functional importance of shedding. Results from our laboratory demonstrated that the concentration of sCD11/CD18 is elevated in synovial fluid as consequence inflammatory arthritis while such fluid under conditions with less or no inflammation has a significantly lower concentration sCD11/CD18 [22]. Systemic alterations, i.e., in the plasma concentration, were also found in inflamed patients compared with healthy volunteers. Although the distribution of the four alpha chains in these complexes is unclear, CD11a was easily detectable in complexes found in human plasma while, by contrast, CD11b was barely detectable and CD11c was not detected. However, in addition to the relative concentration of these species, this finding may at least in part reflect properties of the monoclonal antibodies utilized for detection as well. Other investigations presented evidence that proteolytic cleavage of the extracellular domain serves as an important mechanism for leukocyte detachment after initial cell adhesion and transmigration in both humans and mice [23]. Kinetic studies showed that the cleavage of CD11b is positively correlated with polymorphonuclear leukocyte detachment and subsequent transmigration [24–27].
Detection of Soluble CR3 (CD11b/CD18) by Time-Resolved Immunofluorometry
357
Cross-immunoprecipitation using CD11b and CD18 antibodies further suggested that the cleaved CD11b and CD18 fragments were at least partially associated with one another, suggesting a potential biological function for the soluble integrin fragments [23]. Specific cleavage of the CD18 ectodomain by MMP-9 further supports a functional importance of soluble β2 integrins [28]. Likewise, specific anti-CD11b antibodies that recognize the I-domain are applicable for detecting the soluble ectodomain suggesting that these possess a ligand binding activity [23]. Indeed, in humans sCD11/CD18 was found to be able to bind ICAM-1 immobilized on plastic surfaces, although in this case the binding was most likely contributed by sCD11a/CD18 [22]. In light of the above-described findings and the possible functional importance of CR3 ectodomain shedding in different conditions, we have developed a time-resolved immunofluorometric assay (TRIFMA) to detect sCD11b and sCD18 in serum of both human and murine origin (Fig. 1). The TRIFMA assay is a method for detection of specific analytes very similar to ELISA but with prominent advantages. The long-lasting fluorescence of the europium ions is measured by photon counting with better sensitivity, dynamic range, and reproducibility as compared to the enzymatic based reaction of ELISAs. In addition, the fluorescence emitted from the europium ions upon reading can be measured following storage of the microtiter plates at 4 °C for several days. We believe that these assays may add new tools for understanding the role of cell adhesion molecules in human disease and animal models involving inflammation, notably those involving CR3.
2
Materials All buffers and solutions are made with analytic grade reagents and ultrapure water prepared by purifying deionized water to attain a resistivity of 18 MΩ cm at 25 °C. Buffers are typically supplemented with 10 mM NaN3 to preserve sterility and are stored at 4 °C until use. 1. 96-Well microtiter plates, e.g., FluoroNunc Maxisorp™ microtiter plates (see Note 1). 2. Plastic container. 3. Paper towels. 4. Coating antibody: Monoclonal (m) antibody (Ab) anti-CD11b (e.g., mAb anti-mouse CD11b, clone 5c6) or anti-CD18 antibodies of choice (mAbs, anti-human CD18 antibody, clone KIM185, and anti-mouse CD18 antibody, clone 313903) (see Table 1). 5. Detection antibody: Biotin-labeled mAb anti-CD11b-antibody (e.g., mAb anti-mouse CD11b antibody, clone M1/70.15) or
Gitte Krogh Nielsen and Thomas Vorup-Jensen
a
600
400
200
0
100
murine sCD18 human sCD18
80 60 40 20
50
25
.5 12
5
25 6.
3
12 3.
1
56 1.
1
0.
78
5
39
09 0.
0.
8
0
0.
b
murine sCD11b
19
sCD11b (counts per sec x 10-3)
800
sCD18 (counts per sec x 10-3)
358
Serum conc. (%, v/v)
Fig. 1 Detection of sCD11b and sCD18 by TRIFMA. Serum samples from either male C57BL/6 mice or human volunteers were titrated as indicated in the lower abscissa axis (in serum volume percentage). (a) Serum samples from male C57BL7/6 mice were applied to wells coated with rat mAb anti-mouse CD11b antibody (clone 5c6) followed by development with biotinylated mAb anti-mouse CD11b antibody (clone M1/70.15). (b) Serum samples from human volunteers were applied to wells coated with KIM185 and developed with biotinylated antibody KIM127. Rat mAb anti-mouse CD18 antibody (clone 313903) was used to coat wells to detect CD18 in serum from male C57BL/6 mice, whereas the biotinylated rat mAb anti-mouse CD18 antibody (clone C71/16) was used for development
biotin-labeled monoclonal anti-CD18-antibody of choice (e.g., mAb anti-human CD18 antibody, clone KIM127, and anti-mouse CD18 antibody, clone C71/16) (see Table 1). 6. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.5 mM KH2PO4, 6.9 mM K2HPO4⋅3H2O, pH 7.4. 7. Tris-buffered saline (TBS): 10 mM Tris–HCl, pH 7.4, 145 mM NaCl, 15 mM NaN3.
Thioglycollate-elicited peritoneal macrophages (TPM)
T cell-enriched splenocytes from B10 mice
Monoclonal rat antimouse CD11b (5C6)
Monoclonal rat antimouse CD11b (M1/70.15)
Anti-mouse CD11b
Monoclonal rat anti-mouse Cell membrane glycoproteins from CD18 (C71/16) mouse T-cell lymphoma BW5147
Monoclonal rat antimouse CD18 (313903)
CHO-derived rmIntegrin β2 (Gln24-Asn702) accession # P11835
Human CR3 immunopurified from human white cell lysate. Human CR3 immunized BALB/c mouse spleen cells fused with Sp2/0 cells
Monoclonal mouse anti-human CD18 (KIM127)
Anti-mouse CD18
Human CR3 immunopurified from human white cell lysate. Human CR3 immunized BALB/c mouse spleen cells fused with Sp2/0 cells
Immunogen
Monoclonal mouse anti-human CD18 (KIM185)
Anti-human CD18
Antibody (clone)
IgG2b
Recognizes the murine CD11b cell surface antigen
Recognizes mouse complement type 3 receptor (CR3) and precipitates a heterodimer of 165 and 95 kD
CD18
IgG2a, κ
IgG2b
Integrin beta 2/CD18
Integrin β2/CD18
IgG1, κ
IgG2a
Integrin β2/CD18
Specificity
IgG1, κ
Isotype (Ig class)
Table 1 List of recommended anti-CD11band anti-CD18 monoclonal Abs and their characteristics
Human, mouse, rabbit
Human, mouse
Mouse
Mouse
Human
Human
Reactivity
[32, 33]
[32]
[30, 31]
[29]
[22]
[22]
References
AbD Serotec (Cat# MCA74)
AbD Serotec (Cat# MCA711)
BD Pharmingen (557439)
R&D Systems (MAB2618)
Provider (cat. no.)
359
360
Gitte Krogh Nielsen and Thomas Vorup-Jensen
8. TBS/Tw: TBS with 0.05 % (v/v) Tween-20. 9. Human serum albumin (HSA) (see Note 2). 10. Serum or plasma samples for analysis (store in aliquots at −80 °C until use). 11. Heat-aggregated human IgG (see Note 3). 12. Bovine IgG, rat IgG, and mouse IgG. 13. Assay binding buffer: TBS/tw, 1 mM CaCl2, 1 mM MgCl2, 100 μg/ml heat-aggregated human IgG, 100 μg/ml bovine IgG, 100 μg/ml rat IgG, 100 μg/ml mouse IgG (see Note 4). 14. Biotinylated Ab binding buffer: TBS/Tw, 1 mM CaCl2, 1 mM MgCl2. 15. Streptavidin-Eu3+. 16. TBS/Tw/EDTA: TBS with 0.05 % (v/v) Tween 20 and 25 μM EDTA. 17. Enhancement solution: Mix 5.7 ml of glacial acetic acid with a sufficient amount of 0.1 M potassium hydrogen phthalate to reach pH 3.2. Add 1 ml Triton X-100, and adjust the volume to 1 l with distilled water. Just before use, add 2 μl of a solution containing 15 μM 2-naphthoyltrifluoracetate (2-NTA) and 50 μM tri-o-octylphosphine oxide (TOPO) per ml of the acetic acid/potassium hydrogen phthalate mixture (see Note 5).
3
Methods Carry out all procedures at room temperature unless otherwise specified.
3.1 Detection of Soluble CR3 by TRIFMA
Detection of sCD11b and sCD18 in serum samples is carried out by TRIFMA using a “sandwich” technique with pairs of polystyrenecoated and biotinylated mAbs applied for the detection of either the CD11b chain or the CD18 chain of CR3. 1. Coat the wells of a 96-well microtiter plate by adding the captured mAb diluted in PBS buffer to each well using a total volume of 100 μl per well. The mAb anti-mouse CD11b (clone 5c6) is used in a concentration of 0.5 μg per ml PBS, whereas the optimal concentration of mAb anti-mouse CD18 (clone 313903) is 1 μg per ml PBS. In assay for human samples the mAb anti-human CD18 antibody (clone KIM185) is used at a concentration of 1 μg per ml PBS (see Table 1 for information about the antibodies). 2. Incubate at 4 °C overnight in a moist chamber (see Note 6). 3. Empty the wells by tapping the plate on a layer of paper towels, and block nonspecific binding by adding 200 μl of TBS containing 0.05 % (v/v) Tween 20 to each well. Incubate for 1 h at
Detection of Soluble CR3 (CD11b/CD18) by Time-Resolved Immunofluorometry
361
20 °C. After incubation, empty wells and wash three times with 200 μl of TBS containing 0.05 % (v/v) Tween 20 (see Note 7). 4. Dilute samples of serum (or plasma) 1:10 or 1:20 in binding buffer for antigen assay. Add 100 μl to each well of the coated plate. Incubate in moist chamber overnight at 4 °C. At the end of incubation, empty wells by repeated tapping of the plate on a layer of paper towels, and then wash wells three times with TBS containing 0.05 % (v/v) Tween 20. 5. Dilute the biotinylated mAb in TBS containing 0.05 % (v/v) Tween 20. Rat anti-CD11b antibody (clone M1/70.15) is diluted to 0.25 μg/ml, whereas rat anti-CD18 antibody (clone C71/16) is used at a concentration of 1 μg/ml. The biotinylated mAb anti-human antibody (KIM127) should be used in a concentration of 1 μg/ml. Add 100 μl of the diluted antibody to each well and incubate for 1–2 h at 20 °C in the moist chamber. At the end of incubation, empty wells by tapping the plate repeatedly on a layer of paper towels, and then wash wells three times with TBS containing 0.05 % (v/v) Tween 20. 6. Dilute streptavidin-Eu3+ appropriately (e.g., 1:1,000) in TBS containing 0.05 % (v/v) Tween 20 and 25 μM EDTA. Add 100 μl to each well and incubate for 1–2 h at 20 °C in the moist chamber. At the end of incubation, empty wells by repeated tapping of the plate on a layer of paper towels, and then wash wells three times with TBS containing 0.05 % (v/v) Tween 20. 7. Add 200 μl enhancement solution per well, and incubate plate for 5 min with shaking at room temperature. Read the signals developed in a machine equipped for TRIFMA measurements (see Note 8).
4
Notes 1. For example, FluoroNunc Maxisorp™ microtiter plates with a low background fluorescence. The maxisorp surface is a modified, highly charged polystyrene surface with high affinity to molecules with polar or hydrophilic groups, which implies that when coating with antibodies the hydrophilic Fc region of the antibody is more likely to be bound, thereby leaving the fab region intact for detection of a specific analyte. 2. HSA can be added to the TBS/TW buffer used as blocking buffer to reduce background fluorescence signal. When necessary, we use HSA at a concentration of 1 mg/ml to reduce background fluorescence noise signal. 3. The IgG is heat-aggregated prior to use in the assay by incubation of TBS with 10 mg/ml IgG at 63 °C for 30 min followed
362
Gitte Krogh Nielsen and Thomas Vorup-Jensen
by centrifugation for 10 min at 3,000 × g to remove insoluble complexes (grossly aggregated IgG). The supernatant is then used. Addition of heat-aggregated IgG reduces interference by rheumatoid factor (RF) in the assay. 4. Since immunoglobulin-reactive antibodies are found in sera from humans as well as other species, the inclusion of aggregated human IgG and IgGs from mouse, rat, and cow is a mean of avoiding an influence of the immunoglobulins binding to antibodies applied for antigen detection. The presence of these immunoglobulin-reactive antibodies may cause a false-positive signal through the binding and capture of the antibodies applied for antigen detection. We find that it is best to prepare the assay binding buffer fresh each time. 5. Store both the acetic acid/potassium hydrogen phthalate solution and the NTA/TOPO solutions up to 3 months at 4 °C. Once the solutions are mixed, the complete enhancement solution should be used within a month. Furthermore, it is advisable to check the background fluorescence signal of the enhancement buffer just after preparation. Counts per second should not exceed 1,000. 6. The humid chamber consists of a closed plastic container with a moist paper towel at the bottom. 7. If the plates are to be stored for later use, add 200 μl TBS per well and store at 4 °C in a moist chamber. 8. Measurements of TRIFMA signals can be done using a Victor® plate reader available from PerkinElmer.
Acknowledgements We would like to thank Drs. Jens C. Jensenius and Steffen Thiel, Dept. of Biomedicine, Aarhus University, for their support and excellent advice on applications of TRIFMA. This work was financially supported by grants from the Lundbeck Foundation (LUNA), the Danish Multiple Sclerosis Association, and the Danish Council for Independent Research | Medical Sciences. References 1. Arnaout MA (1990) Structure and function of the leukocyte adhesion molecules CD11/ CD18. Blood 75(5):1037–1050 2. Hibbs ML, Wardlaw AJ, Stacker SA, Anderson DC, Lee A, Roberts TM, Springer TA (1990) Transfection of cells from patients with leukocyte adhesion deficiency with an integrin beta subunit (CD18) restores lymphocyte functionassociated antigen-1 expression and function. J Clin Invest 85(3):674–681
3. Wardlaw AJ, Hibbs ML, Stacker SA, Springer TA (1990) Distinct mutations in two patients with leukocyte adhesion deficiency and their functional correlates. J Exp Med 172(1):335–345 4. Kishimoto TK, Hollander N, Roberts TM, Anderson DC, Springer TA (1987) Heterogeneous mutations in the beta subunit common to the LFA-1, Mac-1, and p150,95 glycoproteins cause leukocyte adhesion deficiency. Cell 50(2):193–202
Detection of Soluble CR3 (CD11b/CD18) by Time-Resolved Immunofluorometry 5. Anderson DC, Springer TA (1987) Leukocyte adhesion deficiency: an inherited defect in the Mac-1, LFA-1, and p150,95 glycoproteins. Annu Rev Med 38:175–194 6. Dana N, Styrt B, Griffin JD, Todd RF 3rd, Klempner MS, Arnaout MA (1986) Two functional domains in the phagocyte membrane glycoprotein Mo1 identified with monoclonal antibodies. J Immunol 137(10):3259–3263 7. Ross GD, Yount WJ, Walport MJ, Winfield JB, Parker CJ, Fuller CR, Taylor RP, Myones BL, Lachmann PJ (1985) Disease-associated loss of erythrocyte complement receptors (CR1, C3b receptors) in patients with systemic lupus erythematosus and other diseases involving autoantibodies and/or complement activation. J Immunol 135(3):2005–2014 8. Park JY, Arnaout MA, Gupta V (2007) A simple, no-wash cell adhesion-based high-throughput assay for the discovery of small-molecule regulators of the integrin CD11b/CD18. J Biomol Screen 12(3):406–417 9. Lee JO, Rieu P, Arnaout MA, Liddington R (1995) Crystal structure of the A domain from the alpha subunit of integrin CR3 (CD11b/ CD18). Cell 80(4):631–638 10. Beller DI, Springer TA, Schreiber RD (1982) Anti-Mac-1 selectively inhibits the mouse and human type three complement receptor. J Exp Med 156(4):1000–1009 11. Diamond MS, Staunton DE, de Fougerolles AR, Stacker SA, Garcia-Aguilar J, Hibbs ML, Springer TA (1990) ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18). J Cell Biol 111(6 Pt 2):3129–3139 12. Rosen H, Law SK (1990) The leukocyte cell surface receptor(s) for the iC3b product of complement. Curr Top Microbiol Immunol 153:99–122 13. Ross GD, Vetvicka V (1993) CR3 (CD11b, CD18): a phagocyte and NK cell membrane receptor with multiple ligand specificities and functions. Clin Exp Immunol 92(2):181–184 14. Tsuji S, Kaji K, Nagasawa S (1994) Activation of the alternative pathway of human complement by apoptotic human umbilical vein endothelial cells. J Biochem 116(4):794–800 15. Ezekowitz RA (2002) Local opsonization for apoptosis? Nat Immunol 3(6):510–512 16. Ross GD (2000) Regulation of the adhesion versus cytotoxic functions of the Mac-1/CR3/ alphaMbeta2-integrin glycoprotein. Crit Rev Immunol 20(3):197–222 17. Dunne JL, Collins RG, Beaud et al, Ballantyne CM, Ley K (2003) Mac-1, but not LFA-1, uses intercellular adhesion molecule-1 to mediate slow leukocyte rolling in TNF-alpha-induced inflammation. J Immunol 171(11):6105–6111 18. Vorup-Jensen T (2012) On the roles of polyvalent binding in immune recognition: perspectives in the nanoscience of immunology and
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
363
the immune response to nanomedicines. Adv Drug Deliv Rev 64(15):1759–1781 Tan SM (2012) The leucocyte beta2 (CD18) integrins: the structure, functional regulation and signalling properties. Biosci Rep 32(3): 241–269 Vorup-Jensen T, Carman CV, Shimaoka M, Schuck P, Svitel J, Springer TA (2005) Exposure of acidic residues as a danger signal for recognition of fibrinogen and other macromolecules by integrin alphaXbeta2. Proc Natl Acad Sci U S A 102(5):1614–1619 Vorup-Jensen T, Chi L, Gjelstrup LC, Jensen UB, Jewett CA, Xie C, Shimaoka M, Linhardt RJ, Springer TA (2007) Binding between the integrin alphaXbeta2 (CD11c/CD18) and heparin. J Biol Chem 282(42):30869–30877 Gjelstrup LC, Boesen T, Kragstrup TW, Jorgensen A, Klein NJ, Thiel S, Deleuran BW, Vorup-Jensen T (2010) Shedding of large functionally active CD11/CD18 Integrin complexes from leukocyte membranes during synovial inflammation distinguishes three types of arthritis through differential epitope exposure. J Immunol 185(7):4154–4168 Zen K, Guo YL, Li LM, Bian Z, Zhang CY, Liu Y (2011) Cleavage of the CD11b extracellular domain by the leukocyte serprocidins is critical for neutrophil detachment during chemotaxis. Blood 117(18):4885–4894 Evans BJ, McDowall A, Taylor PC, Hogg N, Haskard DO, Landis RC (2006) Shedding of lymphocyte function-associated antigen-1 (LFA-1) in a human inflammatory response. Blood 107(9):3593–3599 Mechtersheimer S, Gutwein P, Agmon-Levin N, Stoeck A, Oleszewski M, Riedle S, Postina R, Fahrenholz F, Fogel M, Lemmon V, Altevogt P (2001) Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins. J Cell Biol 155(4):661–673 Phillipson M, Heit B, Colarusso P, Liu L, Ballantyne CM, Kubes P (2006) Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade. J Exp Med 203(12): 2569–2575 Phillipson M, Heit B, Parsons SA, Petri B, Mullaly SC, Colarusso P, Gower RM, Neely G, Simon SI, Kubes P (2009) Vav1 is essential for mechanotactic crawling and migration of neutrophils out of the inflamed microvasculature. J Immunol 182(11):6870–6878 Vaisar T, Kassim SY, Gomez IG, Green PS, Hargarten S, Gough PJ, Parks WC, Wilson CL, Raines EW, Heinecke JW (2009) MMP-9 sheds the beta2 integrin subunit (CD18) from macrophages. Mol Cell Proteomics 8(5):1044–1060 Gomez IG, Tang J, Wilson CL, Yan W, Heinecke JW, Harlan JM, Raines EW (2012)
364
Gitte Krogh Nielsen and Thomas Vorup-Jensen
Metalloproteinase-mediated shedding of integrin beta2 promotes macrophage efflux from inflammatory sites. J Biol Chem 287(7):4581–4589 30. Springer TA (1990) Adhesion receptors of the immune system. Nature 346(6283):425–434 31. Springer TA (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76(2):301–314 32. Rosen H, Gordon S (1987) Monoclonal antibody to the murine type 3 complement
receptor inhibits adhesion of myelomonocytic cells in vitro and inflammatory cell recruitment in vivo. J Exp Med 166(6): 1685–1701 33. Halle A, Hornung V, Petzold GC, Stewart CR, Monks BG, Reinheckel T, Fitzgerald KA, Latz E, Moore KJ, Golenbock DT (2008) The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nat Immunol 9(8):857–865
