मूल्यांकन करने के लिए एक Monocyte Monolayer परख का उपयोग Fcγ रिसेप्टर की मध्यस्थता Phagocytosis
Summary
The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fcγ receptor (FcγR)-mediated phagocytosis.
Abstract
Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fcγ receptor (FcγR)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express FcγRs (e.g., FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA) that are involved in immune responses. The MMA exploits the mechanism of FcγR-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate FcγRs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out FcγR-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo FcγR-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of FcγR-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.
Introduction
The monocyte monolayer assay (MMA) is an in vitro assay originally developed to better predict blood transfusion outcomes in patients with auto- or alloantibodies to red blood cells (RBCs)1-5. By assessing the effect of anti-RBC antibodies in mediating Fcγ receptor (FcγR)-mediated phagocytosis using this in vitro assay, it is possible to predict the clinical outcome in vivo. Indeed, the MMA has been used successfully to avoid immune destruction of antibody-bound RBCs, despite the transfusion of serologically incompatible blood5. The typical pre-transfusion procedure for compatibility testing, also termed crossmatching, involves serological methods that include typing the patient’s blood for ABO and Rh antigens and screening for the presence of anti-RBC antibodies in the patient6. Blood matched for ABO/Rh is selected, and if antibodies are present, an attempt to identify them is made so that blood for transfusion can be further selected to avoid these antigens. An ideal crossmatch result occurs when all donor blood is serologically compatible with the patient’s blood, which reduces the risk of post-transfusion hemolysis7. However, this system falls short for the small group of patients who have become alloimmunized upon repeated transfusion or pregnancy. These patients produce alloantibodies against specific RBC antigens. Some produce antibodies to antigens of very high frequency in the general population, and thus become progressively more difficult to crossmatch8,9. Adding to the complexity, not all alloantibodies are clinically significant; in other words, the binding of an alloantibody to RBCs detected by a serology test does not necessarily result in hemolysis when antigen-positive, incompatible blood is transfused. The MMA was originally developed to assess the potential clinical significance of serologically incompatible blood in a transfusion setting1-5.
Since extravascular hemolysis of antibody-bound RBCs is known to be mediated by the mononuclear phagocyte system, primary monocytes/macrophages are utilized in the development of diagnostic assays. The first assay to study the interaction of monocytes, RBCs, and antibodies was published in 1975, but the sub-optimal conditions used led only to rosette formation (the binding of RBCs to the periphery of the monocyte), and no phagocytosis was observed10. Significant modifications to the assay were made by several groups, leading to an assay for which the level of phagocytosis of alloantibody-bound RBCs could be correlated to the clinical outcome of hemolysis1-5. Recently, the optimal storage conditions of clinical samples and further optimization of assay conditions were examined to enhance the utility of a clinical MMA crossmatch using autologous patient samples11.
Three other diagnostic techniques have been employed in addition to the MMA in predicting transfusion outcomes: the 51Cr release test, the rosette test, and the chemiluminescence test (CLT). In the 51Cr release test, the patient is injected with 51Cr-labeled donor RBCs, and the half-life of the labeled RBCs is monitored and is predictive of post-transfusion survival or clearance12,13. As this method uses radioactive materials, it is rarely performed anymore. The rosette test involves mixing and incubating monocytes with RBCs and quantifying the level of rosette formation (with no phagocytosis)14. The clinical significance of antibodies in vivo involves active phagocytosis by macrophages found in the spleen and/or the liver; thus, this method does not provide a relevant readout of phagocytosis. The CLT uses luminol to monitor the oxidative burst during monocyte phagocytosis of RBC, since luminol fluoresces blue when oxidized in the phagosome15. This method is good, but contamination by neutrophils can confound the readout. Parallel comparisons have been made to evaluate the sensitivity, practicality, and reproducibility of the four available methods, and both the CLT and MMA were ranked superior16. However, the CLT has been mainly utilized in assessing hemolytic disease of the fetus and newborn (HDFN), and the assay’s optimal pH of 8.0 might compromise the level of phagocytosis11.
In addition to its diagnostic and clinical utility, the MMA has been modified for other research purposes. Indeed, the MMA can not only serve as a functional assay to address discrepancies between serology and biology, it has also been used to retrospectively investigate the cause of hemolysis after intravenous immunoglobulin (IVIG) therapy17. It has also been used to examine the structure-function of chemical inhibitors of FcγR-mediated phagocytosis18-20 and to study the downstream signaling of FcγR-mediated phagocytosis21. In our laboratory, in addition to using a human MMA, we are developing a murine MMA using primary mouse peripheral blood mononuclear cells (PBMCs) and autologous RBCs. The rationale is to screen antibodies that can induce FcγR-mediated phagocytosis as an intermediate to developing an in vivo autoimmune hemolytic anemia (AIHA) mouse model (unpublished data). The various modifications focus on different aspects of the IgG antibody and FcγR interaction that induce phagocytosis.
Protocol
Representative Results
Discussion
एमएमए एक थका देने वाली तकनीक है कि दोनों टिशू कल्चर और माइक्रोस्कोपी में विशेषज्ञता की आवश्यकता है। वहाँ सफलता सुनिश्चित करने के लिए कई महत्वपूर्ण कदम उठाए हैं: 1) एककेंद्रकश्वेतकोशिका monolayer की पीढ़ी; 2) लाल रक्त कोशिकाओं की opsonization, और 3) मैनुअल मात्रा का ठहराव। एककेंद्रकश्वेतकोशिका monolayer चैम्बर स्लाइड करने के लिए बहुत दृढ़ता से पालन नहीं करता है, तो शारीरिक पीएच परख 11 भर में बनाए रखा जाना चाहिए और PBMCs की एक पर्याप्त संख्या वरीयता प्राप्त किया जाना चाहिए। जोरदार pipetting, जो पालन कोशिकाओं परेशान हो सकता है, बचा जाना चाहिए। एक दृष्टिकोण हमेशा हटाने और चैम्बर के एक ही कोने से समाधान जोड़ सकते हैं और यह सुनिश्चित करने के लिए कि गति धीमी और स्थिर है करने के लिए है। इसी तरह, अंतिम चरण धोने के अतिरिक्त लाल रक्त कोशिकाओं को दूर करने के दौरान, आंदोलन धीमी और स्थिर होना चाहिए। यह जबकि अभी भी संयुक्त राष्ट्र के phagocytosed लाल रक्त कोशिकाओं के बहुमत को हटाने monolayer करने के लिए कम से कम अशांति सुनिश्चित करता है। अपर्याप्त धोने contaminating लाल रक्त कोशिकाओं के एक उच्च पृष्ठभूमि, जो मैनुअल योग्यता के रूप में आता है को बढ़ावा मिलेगाntification मुश्किल है। दूसरे, 2 आर आर 2 लाल रक्त कोशिकाओं को पर्याप्त phagocytosis नियंत्रण के लिए 80-120 के एक औसत phagocytic सूचकांक प्राप्त करने के लिए opsonized किया जाना चाहिए। यह वांछित phagocytic रेंज गिनती की आसानी के बीच एक संतुलन बनाता है (उदाहरण के लिए, अधिक से अधिक 5 से monocytes phagocytosed लाल रक्त कोशिकाओं को सही अंदाजा लगाना मुश्किल कर रहे हैं) और सांख्यिकीय विश्लेषण के लिए phagocytosis की एक पर्याप्त राशि को बनाए रखने। opsonization की डिग्री एक आई ए टी द्वारा की पुष्टि की जा सकती है, और 4 + 3 + करने के बीच एक पढ़ने की जरूरत है। 2 आर आर 2 लाल रक्त कोशिकाओं जब वहाँ धोने के दौरान अतिरिक्त सेल है, जब सतह पर तैरनेवाला गहरे लाल रंग बदल जाता है, या खारिज किया जाना चाहिए phagocytosis में एक महत्वपूर्ण कमी भंडारण में कोशिकाओं की उम्र बढ़ने के कारण प्रयोगों में मनाया जाता है। अन्त में, मैनुअल माइक्रोस्कोप का उपयोग मात्रा का ठहराव, खासकर जब प्रयोगशाला कर्मियों के बीच और प्रयोगों के बीच में गिना जाता है की तुलना, मुश्किल हो सकता है। अच्छी तरह से प्रत्येक पर एक ही क्षेत्र की जांच करके, या बस अधिक कोशिकाओं की गणना के द्वाराएक और अधिक सुसंगत गिनती प्राप्त किया जा सकता है। साइड-बाई-साइड एक अनुभवी तकनीशियन और प्रशिक्षण स्लाइड के एक नामित सेट के उपयोग के साथ प्रशिक्षण की सिफारिश की है।
एमएमए के एक प्रमुख आलोचक मैनुअल मात्रा का ठहराव कदम की आत्मीयता है। हालांकि, पर्याप्त प्रशिक्षण के साथ, स्थिरता विभिन्न काउंटरों भर में प्राप्त किया जा सकता है। एक और सीमा 2 आर 2 सतह प्रतिजन अभिव्यक्ति के स्तर है, जो डेटा बदलाव का एक स्रोत है जब मानव नमूनों के साथ काम कर रहा है monocyte phagocytic क्षमताओं में और अनुसंधान में आंतरिक दाता करने वाली दाता मतभेद है।
अन्य वैकल्पिक तकनीकों FcγR की मध्यस्थता phagocytosis की जांच के लिए उपलब्ध हैं। वाणिज्यिक किट के बहुमत phagocytosis की निगरानी के लिए फ्लोरोसेंट उत्पादन का उपयोग (जैसे, bioparticles, पीएच के प्रति संवेदनशील प्रतिदीप्ति प्रोटीन, या आईजीजी लेबल फ्लोरोसेंट लेटेक्स मोती)। फ्लोरोसेंट उत्पादन का उपयोग अधिक उद्देश्य मात्रा का ठहराव की पेशकश करता है, लेकिन एक भी चुनाव करने की जरूरत हैदोबारा उपलब्धता, लागत, और एक फ्लोरोसेंट माइक्रोस्कोप या एक प्रवाह कोशिकामापी, साथ ही व्यावसायिक रूप से उपलब्ध किट पर आगामी निर्भरता के इस्तेमाल से जुड़े प्रशिक्षण।
अंत में, इस परख अनुसंधान प्रश्न के आधार पर संशोधित किया जा सकता है। उदाहरण के लिए, जब phagocytosis की दवा निषेध का परीक्षण, monocytes पूर्व में इलाज किया जा सकता है या तो या दोनों दवाओं और opsonized लाल रक्त कोशिकाओं (यानी, एक प्रतियोगिता परख) के साथ सह-incubated। विभिन्न उपप्रकार, chimeric एंटीबॉडी, या पुनः संयोजक निर्माणों की एंटीबॉडी के बहाव के संकेत भी परीक्षण किया जा सकता है। एक सार्वभौमिक प्रतिजन अशक्त रक्त 24 के विकास में हाल ही में सफलताओं के साथ, एमएमए का आकलन करने के लिए वहाँ वास्तव में phagocytosis ट्रिगर में एक कम प्रभावकारिता है कि क्या विभिन्न एंटीबॉडी के साथ इन प्रतिजन अशक्त लाल रक्त कोशिकाओं की प्रारंभिक स्क्रीन में उपयोग किया जा सकता है।
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank the Canadian Blood Services for a Graduate Fellowship Program Award to T.N.T. This research received financial support from the Canadian Blood Services’ Centre for Innovation, funded by the federal government (Health Canada) and the provincial and territorial ministries of health. The views herein do not reflect the views of the federal, provincial, or territorial governments in Canada.
Materials
| Acid citrate dextrose (ACD) vacutainers | BD | REF364606 | |
| RPMI 1640 | Sigma | R8758 | |
| HEPES | Bioshop | HEP003.100 | |
| Fetal bovine serum | Multicell | 080150 | |
| Gentamicin | Gibco | 15710-64 | |
| Ficoll-Paque PLUS | GE | 17-1440-03 | https://www.gelifesciences.com/ |
| Phosphate buffered saline | Sigma | D8537 | |
| 8-chamber slides | Lab-Tek-ll | 154534 | |
| R2R2 (cDE/cDE) red blood cells | Canadian Blood Services | Commercially available (e.g. http://www.bio-rad.com/en-ca/product/reagent-red-blood-cells) | |
| Polyclonal anti-D from human serum | Gamma Biologics | DIN 02247724 | Can be substituted with commercially available monoclonal anti-D or with Rh immune globulin |
| 100% methanol | Caledon | 6700-1-42 | |
| Polyvinyl alcohol resin | Sigma | P8136 | Can be substituted with commercially available mount |
| UltraPure glycerine | Invitrogen | 15514-011 | |
| Cover slips | VWR | 48366 067 | |
| Novaclone anti-IgG | Immucorgamma | 5461023 | Optional for IAT (http://www.fda.gov/downloads/biologicsbloodvaccines/…/ucm081743.pdf) |
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