Hindawi Journal of Immunology Research Volume 2020, Article ID 6292631, 4 pages https://doi.org/10.1155/2020/6292631 Research Article Anti-HMGCR Specificity of HALIP: A Confirmatory Study Andrés Baucells ,1 Maria Angeles Martínez ,1 Marcelo Alvarado-Cardenas,2 Anaís Mariscal,1 Laura Martinez-Martinez,1 Cándido Juárez ,1 and Albert Selva-O’Callaghan 2 1 Immunology Department, Hospital de la Santa Creu i Sant Pau, Barcelona 08041, Spain 2 Systemic Autoimmune Diseases Unit, Hospital Vall d’Hebrón, Medicine Department, Universidad Autónoma de Barcelona, Barcelona, Spain Correspondence should be addressed to Andrés Baucells; abaucells@santpau.cat Received 28 February 2020; Accepted 25 June 2020 Academic Editor: Giuseppe A. Sautto Copyright © 2020 Andrés Baucells et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A distinctive new indirect immunofluorescence pattern in liver tissue has been associated with anti-HMGCR autoantibodies. It is known as HALIP (HMGCR Associated Liver Immunofluorescence Pattern). In this study, we furthered the original studies to demonstrate the association of anti-HMGCR antibodies with the HALIP. Human anti-HMGCR antibodies from patients’ sera were purified and incubated with rat triple tissue (kidney/stomach/liver). A characteristic HALIP was observed. Additionally, a colocalization assay of human anti-HMGCR antibodies with rabbit polyclonal anti-HMGCR antibodies showed colocalization of both immunofluorescence patterns. This study confirms that the HALIP is due to human anti-HMGCR antibodies. 1. Introduction liver sections associated with anti-HMGCR antibodies. They called it the HALIP (HMGCR Associated Liver IFL Pattern). Idiopathic inflammatory myopathies (IIM), also known as Here, we further identify these autoantibodies as responsible myositis, are a group of heterogeneous disorders character- for the previously described characteristic IIF pattern. Our ized by a distinct inflammatory infiltrate in muscle tissue findings demonstrate that the HALIP is specific for human [1]. Several phenotypes linked to specific autoantibodies have anti-HMGCR antibodies, as shown by the IIF pattern of the been described [1, 2]. One of these phenotypes includes purified human anti-HMGCR antibodies and by the coloca- patients exposed to statins who develop a statin-associated lization of these human autoantibodies with rabbit poly- autoimmune myopathy with antibodies against the pharma- clonal anti-HMGCR antibodies. cologic target of statins, the 3-hydroxy-3-methylglutaryl- coenzyme A reductase (HMGCR). These autoantibodies and their link to a specific myositis phenotype were described 2. Material and Methods in 2010 by Mammen et al. [3]. The pathological characteris- 2.1. Material tics of this statin autoimmune myopathy are those of the immune-mediated necrotizing myopathy (IMNM), defined 2.1.1. Sera. The human sera used in the assay were obtained by the presence of necrotic fibres found in muscle biopsy. from 3 patients with statin-associated necrotizing myopathy Severe proximal muscle weakness and high levels of creati- that tested positive with a high titre of anti-HMGCR anti- nine kinase are also hallmarks of the disease [4, 5]. bodies by ELISA (INOVA Diagnostics, San Diego, CA). In 2016, Alvarado-Cardenas et al. [6] described a distinc- Also, three healthy human sera were used as negative con- tive, novel indirect immunofluorescence (IIF) pattern on rat trols. The diagnostic criteria for IMNM were based on the 2 Journal of Immunology Research IMNM diagnostic and classification criteria proposed by the 3. Results Muscle Study Group/European NeuroMuscular Centre (MSG/ENMC) [7]. 3.1. Immunofluorescence Pattern of the Purified Human Anti- HMGCR Antibodies. The anti-HMGCR patients’ positive sera stain isolated hepatocytes located periportally on rat 2.1.2. IIF Assays. All the IIF assays were performed on rat tri- liver, as reported for the HALIP (Figure 1(a)). Rat triple tis- ple tissue INOVA slides according to the instructions of the sue was also incubated with normal human sera as a negative NOVA Lite Rat Liver, Kidney, Stomach Kit (INOVA Diag- control (Figure 1(b)). As shown in Figure 1(c), the HALIP nostics, San Diego, CA). disappeared after absorption of anti-HMGCR autoanti- bodies with recombinant HMGCR, supporting that specific 2.2. Purification of Human Anti-HMGCR Antibodies. 5 μg of anti-HMGCR antibodies are responsible for the pattern. recombinant HMGCR protein (Origene, Rockville, MD) was This conclusion is further confirmed by the studies per- run on SDS-gel electrophoresis and transferred into a nitro- formed with purified antibodies. In fact, as observed in cellulose membrane as previously described [8]. Briefly, we Figure 1(d), the IIF pattern on rat liver tissue of the purified blocked the membrane with 5% casein in PBS and then cut anti-HMGCR antibodies reproduces the same pattern of the two vertical strips at the ends of the membrane. The strips anti-HMGCR patients’ positive sera on Figure 1(a). The were then incubated with the human anti-HMGCR serum purified negative control showed no staining on triple tissue and, following the blot protocol [8], the position of the pro- (data not shown), thus confirming that the purification of tein was localized. Next, we incubated the remaining section the HMGCR autoantibodies is specific. of the membrane overnight, covered with 6 ml of anti- HMGCR patient serum dilution (diluted 1/20 in 3% casein 3.2. Immunofluorescence Pattern of the Colocalization Assay. in PBS). We then washed the membrane three times with The results obtained above indicate that the differential PBS 0.01% tween and cut a horizontal strip off the membrane expression of HMGCR in rat liver cells is responsible for horizontally 0.3 cm above and 0.3 cm below the localized pro- the HALIP. A commercial antibody obtained by immuniza- tein. On continuation, we cut the strip into little pieces and tion with HMGCR would therefore reproduce the HALIP, placed them in a tube. recognizing the same hepatocytes as those in patients’ sera. At this point, we eluted the antibodies bound to the To demonstrate this, we incubated the rat triple tissue with pieces with 250 μl of glycine pH = 2, for 2 minutes under agi- both human and commercially obtained rabbit anti- tation. The elute was then buffered with 25 μl of Tris pH = 9:5 HMGCR antibodies. As expected, we observed that the to take it back to pH = 7. Finally, we incubated 50 μl of the human anti-HMGCR antibodies (Figure 2(a)) stain exactly buffered elute in rat tissue following the IIF INOVA kit the same rat liver cells as the rabbit polyclonal anti-HMGCR instructions. antibodies (Figure 2(b)). This is clearly shown in the merged A strip was cut from the remaining part of the membrane image (Figure 2(c)) that confirms the correspondence of the and incubated with the HMGCR antibodies following the two stainings. same protocol described above. It was used as a purification negative control. 4. Discussion 2.3. Colocalization of Human and Rabbit Polyclonal Anti- In this study, using purified human anti-HMGCR antibodies HMGCR Antibodies. First, we incubated the rat tissue with and colocalization studies, we demonstrate that the HALIP is 50 μl of anti-HMGCR Rabbit Polyclonal Antibody (1/50 due to anti-HMGCR antibodies. dilution in PBS) (Invitrogen, Frederick, MD) for 1 h in a The fact that purified human anti-HMGCR antibodies humid chamber. After washing thrice with PBS, the tissue are directed against some rat liver cells is not surprising was incubated with goat anti-rabbit IgG (H+L) labelled with because hepatocytes are responsible for the synthesis of 60- Alexa Fluor 594 (Invitrogen, Frederick, MD) for 1 h. We then 70% of the cholesterol for the whole body [9]. HMGCR is incubated the tissue with the anti-HMGCR-positive human mainly expressed in periportal hepatocytes, as reflected by serum (1/80 in PBS) for 30 min, followed by washing and the HALIP. This distribution was previously reported in incubation with a drop of goat anti-human FITC IgG H&L studies that analysed the effects of statin on the liver [10, conjugate (from the triple tissue INOVA kit) for another 11]. These studies show that the initial expression of 30 min. HMGCR in periportal hepatocytes changes drastically to a Each of the conjugates was tested individually to ensure more generalized expression in rat liver after statin treat- there was no crosspositivity between the excitability wave- ment. As rat and human HMGCR share 93% homology, length of the two different fluorochromes. the rat triple tissue is a good substratum to test for human anti-HMGCR autoantibodies [6]. Though HMGCR is pres- 2.4. Absorption of the Anti-HMGCR Antibodies. We incu- ent in the liver, anti-HMGCR antibodies are associated with bated the serum of a patient with anti-HMGCR antibodies myositis. To our knowledge, only one previous case of auto- (1/80 dilution in PBS) with an excess of HMGCR recombi- immune hepatitis and anti-HMGCR statin-associated auto- nant protein (Sigma-Aldrich, St Louis, MO) at a concentra- immune myositis patient has been reported to date [12, 13]. tion of 25 μg/ml for 30 minutes. The absorbed serum was As IIF is a common and inexpensive screening technique then poured on rat sections to test for the HALIP. available in most clinical laboratories, the HALIP can be Journal of Immunology Research 3 (a) (b) (c) (d) Figure 1: The figures portray a representative image of one of the anti-HMGCR human sera staining patterns on rat liver. (a) HALIP as described, staining some isolated hepatocytes on liver rat sections after incubation with an anti-HMGCR-positive human serum (dilution 1/80). (b) Normal human serum as negative control (dilution 1/80). (c) Anti-HMGCR-positive serum after absorption with HMGCR recombinant protein (dilution 1/80). (d) HALIP staining pattern after incubation with antibodies eluted from gel-fractionated recombinant HMGCR protein. (a) (b) (c) Figure 2: HALIP of (a) a human anti-HMGCR-positive sera conjugated with FITC, (b) polyclonal rabbit anti-HMGCR sera conjugated with Alexa 594, and (c) the merging of (a) and (b). extremely useful for laboratory analysis. Rat triple tissue 2/23 anti-HMGCR-positive myositis patients, according to could be used as an initial screening test for anti-HMGCR Alvarado-Cardenas et al. [6]. The reason for this discordance antibodies in laboratories where the specific antigen detec- remains to be assessed. tion assay is not available. Moreover, if a HALIP is observed In conclusion, our findings in this report confirm that in our routine work as an unexpected finding, the presence of anti-HMGCR antibodies are responsible for the HALIP on anti-HMGCR antibodies should be tested. rat liver. Despite the specificity of the HALIP, confirmation with an antigen detection assay is strongly recommended, as we have observed similar patterns that were negative to anti- Data Availability HMGCR antibodies in the confirmation test. Furthermore, a negative HALIP does not rule out the presence of anti- The data used to support the findings of this study are HMGCR antibodies, because the HALIP was not found in included within the article. 4 Journal of Immunology Research Conflicts of Interest the rat liver and intestine is affected by cholestyramine and mevinolin,” Journal of Lipid Research, vol. 29, no. 6, pp. 781– The authors declare that there is no conflict of interest 796, 1988. regarding the publication of this paper. [12] D. Palamuthusingam, M. Mantha, and S. Dheda, “HMG CoA reductase inhibitor associated myositis and autoimmune hep- Acknowledgments atitis,” Internal Medicine Journal, vol. 47, no. 10, pp. 1213– 1215, 2017. 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