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Novel autoantibodies in Sjogren's syndrome
Clinical Immunology (2012) 145, 251–255
available at www.sciencedirect.com
Clinical Immunology www.elsevier.com/locate/yclim
BRIEF COMMUNICATION
Novel autoantibodies in Sjogren's syndrome Long Shen a , Lakshmanan Suresh b , Matthew Lindemann b , Jingxiu Xuan c , Przemek Kowal b , Kishore Malyavantham b , Julian L. Ambrus Jr. a,⁎ a
Division of Allergy, Immunology and Rheumatology, Department of Medicine, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA b IMMCO Diagnostics Inc., Buffalo, NY 14228, USA c Division of Nephrology, Department of Medicine, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA
Received 21 April 2012; accepted with revision 30 September 2012 Available online 12 October 2012
KEYWORDS Sjogren's syndrome; Autoantibodies
Abstract Sjogren's syndrome (SS) is defined by autoantibodies to Ro and La. The current studies identified additional autoantibodies in SS to salivary gland protein 1 (SP-1), carbonic anhydrase 6 (CA6) and parotid secretory protein (PSP). These autoantibodies were present in two animal models for SS and occurred earlier in the course of the disease than antibodies to Ro or La. Patients with SS also produced antibodies to SP-1, CA6 and PSP. These antibodies were found in 45% of patients meeting the criteria for SS who lacked antibodies to Ro or La. Furthermore, in patients with idiopathic xerostomia and xerophthalmia for less than 2 years, 76% had antibodies to SP-1 and/or CA6 while only 31% had antibodies to Ro or La. Antibodies to SP-1, CA6 and PSP may be useful markers for identifying patients with SS at early stages of the disease or those that lack antibodies to either Ro or La. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Sjogren's disease (SD) is a systemic autoimmune disease in which loss of salivary gland and lachrymal gland function is associated with hypergammaglobulinemia, autoantibody production, mild kidney and lung disease and eventually lymphoma [1–3]. Sjogren's syndrome (SS) involves dry eyes and dry mouth without ⁎ Corresponding author at: Division of Allergy, Immunology and Rheumatology, SUNY at Buffalo School of Medicine and Biomedical Sciences, Room C281, Buffalo General Hospital, 100 High Street, Buffalo, NY 14203, USA. Fax: + 1 716 859 1249. E-mail address: [email protected] (J.L. Ambrus). 1521-6616/$ - see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clim.2012.09.013
systemic features that may be either primary or secondary to another autoimmune disease, such as SLE. Patients with SS and SD are generally picked up at a late stage in their disease, after the salivary glands and lachrymal glands are already destroyed, because they are asymptomatic until that time. At this point, only symptomatic treatment can be offered for abnormal lachrymal and salivary gland function [4–6]. The current diagnostic criteria for SS based on revised American–European consensus group include I. Ocular symptoms, II. Oral symptoms, III. Ocular signs, IV. Focal sialoadenitis, V. Salivary gland involvement and VI. Anti Ro/La antibodies in the absence of head and neck radiation treatment, hepatitis C, AIDS, lymphoma, sarcoidosis, graft versus host disease or
252 anticholinergic drugs. Diagnosis is based on meeting 3 of 4 objective criteria or 4 of 6 total criteria [7]. Many other autoantibodies have been associated with SS and SD including anti-muscarinic receptor 3, anti-tissue kallikrein, anti-α-fodrin and anti-carbonic anhydrase II, but the significance of these autoantibodies has not been fully appreciated [8–15]. None of them are currently included in the diagnostic criteria for SS. We have undertaken studies to investigate early events in the development of SS and SD. Because patients are not identified during early SS and SD, we have utilized animal models to investigate this aspect of the disease. We have developed the interleukin 14 alpha transgenic mouse (IL14αTG) that develops many of the features of SD in the same relative time frame as patients, hypergammaglobulinemia, autoantibodies, loss of salivary gland function, infiltration of salivary and lachrymal glands with lymphocytes, lymphocytic interstitial pneumonia, mild renal disease and eventually lymphoma [16–18]. Findings from this model have been corroborated in the NOD mouse, another model for SS, and in patients with SS and SD when possible [17,18]. The current studies investigate the expression of novel autoantibodies that were first identified in IL14αTG mice but subsequently shown to be present in NOD mice and in patients with SS and SD.
2. Materials and methods 2.1. Serum samples Sera were collected from mice using a protocol approved by the University at Buffalo IACUC committee (MED17105). Sera were collected from patients and normal controls by a protocol approved by University at Buffalo IRB committee (MED60510410E).
2.2. Determination of autoantibodies for SP-1, CA6, PSP, Ro and La Western blot were performed to evaluate the presence of autoantibodies for murine SP-1, CA6 and PSP following procedure described in previous studies [18]. ELISA's were performed to evaluate the presence of autoantibodies for murine Ro and La, human CA6 and PSP following procedure described in previous studies [16]. Antibodies to the human Ro and La were determined by ImmcoStripe Line Immunoassay for anti-nuclear antibody (Immco Diagnostics, Buffalo, NY, USA). The line Immunoblot assay consisted of immobilized auto-antigens (Ro52, Ro60 and La) and reacted as per the product protocol.
3. Results Previous studies recognized that the expression of Ro and La mRNA is increased in patients with SS and SD who have antibodies to Ro and La [19]. We observed that the mRNA for several salivary gland proteins, salivary protein 1 (SP-1), parotid secretory protein (PSP) and carbonic anhydrase 6 (CA6) was expressed at much higher levels in the mRNA of IL14αTG mice compared to control C57BL/6 mice. We therefore examined whether these proteins were target autoantigens in the SD of these mice. At the same time, we
L. Shen et al. examined whether the NOD mouse, which gets similar injury to the salivary and lachrymal glands as the IL14αTG mouse, produced autoantibodies to the antigens. Fig. 1A demonstrates that both IL14αTG mice as well as NOD mice produced autoantibodies recognizing SP-1, PSP and CA6. The control C57BL/6 mice did not make these autoantibodies. Both male and female IL14αTG mice produced these autoantibodies and mice that had antibodies to SP-1 generally also had antibodies to CA6 and PSP as well. Of 6 IL14αTG mice studied, 6 had antibodies to SP-1, 6 had antibodies to CA6 and 5 had antibodies to PSP. Similarly for 6 NOD mice studied, 6 had antibodies to SP-1, 6 had antibodies to CA6 and 4 had antibodies to PSP. Of 6 C57/BL6 control mice studied, none of them had antibodies to SP-1, CA6 or PSP. While antibodies to Ro and La are used as diagnostic criteria for SS and SD, we noted that only 25% of IL14αTG mice developed these autoantibodies. We therefore examined the time course of production of antibodies to Ro, La, SP-1 and CA6 in a cohort of IL14αTG mice. We have previously observed that IL14αTG mice have decreased salivary flow at 6 months of age with autoantibody deposition in the salivary glands. By 10 months of age IL14αTG mice have lymphocytic infiltration of the submandibular glands and by 14 months of age lymphocytic infiltration of the parotid glands as well [17]. As shown in Fig. 1B, antibodies to SP-1 and CA6 were present in more than 50% of IL14αTG mice by 6 months of age and present in all of the mice by 14 months of age. In contrast, none of the IL14αTG mice had antibodies to Ro or La at 6 months of age. By 14 months of age, 25% of IL14αTG mice had these autoantibodies. We next examined whether patients with SS and SD had antibodies to SP-1, CA6 and PSP. We evaluated autoantibodies to CA6 and PSP by ELISA assays using the human versions of these proteins. There is no human equivalent for SP-1, so we utilized a Western blot assay for the presence of this autoantibody using the murine protein. Fig. 2A demonstrates recognition of SP-1 by the sera of a patient with SD, but not the sera of a normal control. Next we evaluated the sera of 13 patients who met diagnostic criteria for SS and had disease for more than 5 years. Fig. 2B demonstrates that 62% of these patients had antibodies to Ro or La, 54% had antibodies to SP-1, 54% had antibodies to CA6 and 69% had antibodies to SP-1 or CA6. Only 20% of patients had antibodies to only CA6 or SP-1. In this cohort 18% had antibodies to PSP and 38% lacked antibodies to either Ro or La. All the patients had one of these autoantibodies. Evaluating a sex and age matched cohort of normal controls (Fig. 2C), 4.3% had antibodies to Ro or La, 4.3% had antibodies to SP-1 3% had antibodies to CA6 and 10% had antibodies to PSP. Using unpaired student's t test, the difference in Ro/La positivity (p = .0093) or SP-1/CA6 positivity (p = .0005) between SS patients and normal controls was highly significant. The difference in Ro/La positivity versus SP-1/CA6 positivity in SS patients was not significant (p = .400). Evaluating 10 patients with rheumatoid arthritis, but not secondary SS, none had antibodies to SP-1, while one had antibody to CA6, one to Ro, two to La and one to PSP (Fig. 2D). These patients were selected to be both RF positive (50%) and RF negative (50%). Because antibodies to SP-1 and CA6 occurred at an earlier time point in IL14αTG mice than antibodies to Ro and La, we also investigated a cohort of 29 patients who had idiopathic
Novel autoantibodies in Sjogren's syndrome
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Figure 1 A. Evaluation of antibodies to SP-1, CA6 and PSP by Western blot in sera of IL14aTG mice, NOD mice and C57BL/6 mice; representative of 6 mice studied per group. B. Time course of antibodies to SP-1, CA6, Ro and La in the sera of 6 IL14αTG mice.
xerostomia and xerophthalmia for less than 2 years. These patients met at least 3 diagnostic criteria for SS and all the exclusion criteria. Fig. 2E demonstrates that of these patients, 31% had antibodies to Ro or La while 76% had antibodies to SP-1 or CA6. In this cohort, 45% had antibodies only to SP-1, 3% had antibodies to only CA6, no patients had antibodies only to Ro/La and 21% had none of these autoantibodies. In this cohort of patients, using unpaired student's t test, the difference in Ro/La positivity (p = .0187) or SP-1/CA6 positivity (p b .0001) between SS patients and normal controls was highly significant. The difference in Ro/La positivity versus SP-1/CA6 positivity in SS patients was also highly significant (p = .0004). Finally, we investigated 20 patients who met full diagnostic criteria for SS with positive salivary gland biopsies, but lacked antibodies to Ro or La. As shown in Fig. 2F, 45% of these patients had antibodies to SP-1 while 5% had antibodies to CA6.
4. Discussion The current studies demonstrate the presence of autoantibodies to SP-1, PSP and CA6 in two animal models of SS and in patients with SS. These autoantibodies occur earlier than antibodies to Ro and La and are present in patients who meet diagnostic criteria for SS but do not have antibodies to either Ro or La. SP-1 antibodies have the greatest specificity and
sensitivity for early SS. Autoantibodies to CA6 add additional sensitivity to the diagnosis of early SS along with SP-1, Ro and La. Carbonic anhydrase 6 is an enzyme involved with the buffering capacity of saliva that is found in both the cytoplasm and the secretory granules of the serous acinar cells in the submandibular and parotid glands [20]. Previous studies have demonstrated antibodies to CA6 in patients with SS, but the significance or timing of the production of these autoantibodies was not pursued [15]. In these studies we demonstrate that antibodies to CA6 occur very early in the course of SD. They occur rarely in normal controls or patients with rheumatoid arthritis lacking xerostomia and xerophthalmia. They rarely occur in patients who do not also have antibodies to SP-1. Parotid secretory protein is a secreted protein that is involved with binding and helping to clear various infectious agents [21,22]. It could easily become an autoantigen as it is bound to foreign proteins and cleaved by local proteases creating neoantigens. Interestingly mice and humans making antibodies to PSP generally make antibodies to CA6 at the same time. Antibodies to PSP occur only rarely in normal controls or patients with rheumatoid arthritis. Further study will be needed to determine whether testing for antibodies to PSP will increase the sensitivity of diagnosis for SS when antibodies to SP-1 and CA6 are already being done. Salivary protein 1 was identified in mice as an mRNA expressed at high levels in the submandibular glands and
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Figure 2 A. Western blot study showing antibodies to SP-1, CA6 and PSP in a representative patient with Sjogren's syndrome and normal control. This figure is representative of over 20 patients and normal controls studied in each group. B. Evaluations of antibodies to Ro, La, SP-1, CA6 and PSP in 13 patients with Sjogren's disease for more than 5 years. C. Evaluation of antibodies to SP-1, CA6, PSP, Ro and La in the sera of 23 normal controls. D. Evaluation of antibodies to SP-1, CA6, PSP, Ro and La in 10 patients with rheumatoid arthritis but not secondary Sjogren's syndrome. E. Evaluation of antibodies to SP-1, CA6, PSP, Ro and La in 29 patients with idiopathic xerostomia and xerophthalmia for less than 2 years. F. Evaluation of antibodies to CA6 and SP-1 in the sera of 20 patients with Sjogren's syndrome lacking antibodies to either Ro or La.
lachrymal glands of mice [23]. It was identified as one of the genes upregulated in the thymus by the autoimmune regulator gene (AIRE) to prevent development of T lymphocytes reactive to the salivary glands [24]. The function of the protein is unknown. The human homologue to SP-1 is also unknown. Interestingly there is a high degree of homology between SP-1 and a putative lipoprotein of Clostridium perfringens (gb EDT 72214.1). It is possible that reactivity to SP-1 occurs because of cross-reactivity to proteins produced by normal or abnormal flora in the respiratory and/or gastrointestinal tract. As a marker for SD, antibodies to SP-1 occur very early in the course of disease and occur in mouse models of SD and patients with SS and SD independent of antibodies to CA6, PSP, Ro or La. They have high sensitivity for SS and SD and are rarely found in patients with rheumatoid arthritis lacking secondary SS. Antibodies to SP-1 were found in 4.3% of normal controls, but it is unclear how many of these “normal controls” may have had early SS. The diagnostic criteria for SS and SD are undergoing continuous revision as more is learned about the pathophysiology of
these disorders. While Ro and La are autoantigens included in the diagnostic criteria for SS, many studies have identified patients with autoimmune SS and SD that do not have these autoantibodies [25–28]. Other autoantibodies such as the ones described in this manuscript may identify other subsets of patients and/or patients at different stages of the disease. One of the current challenges in SS and SD is to identify patients early enough so that therapy can result in recovery of their salivary and lachrymal glands. The autoantibodies to SP-1, CA6 and PSP may be helpful in this regard. New concepts regarding early events in SS and SD may provide therapeutic targets once these patients are identified [3,18].
Conflict of interest statement The authors declare that there are no conflicts of interest.
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available at www.sciencedirect.com
Clinical Immunology www.elsevier.com/locate/yclim
BRIEF COMMUNICATION
Novel autoantibodies in Sjogren's syndrome Long Shen a , Lakshmanan Suresh b , Matthew Lindemann b , Jingxiu Xuan c , Przemek Kowal b , Kishore Malyavantham b , Julian L. Ambrus Jr. a,⁎ a
Division of Allergy, Immunology and Rheumatology, Department of Medicine, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA b IMMCO Diagnostics Inc., Buffalo, NY 14228, USA c Division of Nephrology, Department of Medicine, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA
Received 21 April 2012; accepted with revision 30 September 2012 Available online 12 October 2012
KEYWORDS Sjogren's syndrome; Autoantibodies
Abstract Sjogren's syndrome (SS) is defined by autoantibodies to Ro and La. The current studies identified additional autoantibodies in SS to salivary gland protein 1 (SP-1), carbonic anhydrase 6 (CA6) and parotid secretory protein (PSP). These autoantibodies were present in two animal models for SS and occurred earlier in the course of the disease than antibodies to Ro or La. Patients with SS also produced antibodies to SP-1, CA6 and PSP. These antibodies were found in 45% of patients meeting the criteria for SS who lacked antibodies to Ro or La. Furthermore, in patients with idiopathic xerostomia and xerophthalmia for less than 2 years, 76% had antibodies to SP-1 and/or CA6 while only 31% had antibodies to Ro or La. Antibodies to SP-1, CA6 and PSP may be useful markers for identifying patients with SS at early stages of the disease or those that lack antibodies to either Ro or La. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Sjogren's disease (SD) is a systemic autoimmune disease in which loss of salivary gland and lachrymal gland function is associated with hypergammaglobulinemia, autoantibody production, mild kidney and lung disease and eventually lymphoma [1–3]. Sjogren's syndrome (SS) involves dry eyes and dry mouth without ⁎ Corresponding author at: Division of Allergy, Immunology and Rheumatology, SUNY at Buffalo School of Medicine and Biomedical Sciences, Room C281, Buffalo General Hospital, 100 High Street, Buffalo, NY 14203, USA. Fax: + 1 716 859 1249. E-mail address: [email protected] (J.L. Ambrus). 1521-6616/$ - see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clim.2012.09.013
systemic features that may be either primary or secondary to another autoimmune disease, such as SLE. Patients with SS and SD are generally picked up at a late stage in their disease, after the salivary glands and lachrymal glands are already destroyed, because they are asymptomatic until that time. At this point, only symptomatic treatment can be offered for abnormal lachrymal and salivary gland function [4–6]. The current diagnostic criteria for SS based on revised American–European consensus group include I. Ocular symptoms, II. Oral symptoms, III. Ocular signs, IV. Focal sialoadenitis, V. Salivary gland involvement and VI. Anti Ro/La antibodies in the absence of head and neck radiation treatment, hepatitis C, AIDS, lymphoma, sarcoidosis, graft versus host disease or
252 anticholinergic drugs. Diagnosis is based on meeting 3 of 4 objective criteria or 4 of 6 total criteria [7]. Many other autoantibodies have been associated with SS and SD including anti-muscarinic receptor 3, anti-tissue kallikrein, anti-α-fodrin and anti-carbonic anhydrase II, but the significance of these autoantibodies has not been fully appreciated [8–15]. None of them are currently included in the diagnostic criteria for SS. We have undertaken studies to investigate early events in the development of SS and SD. Because patients are not identified during early SS and SD, we have utilized animal models to investigate this aspect of the disease. We have developed the interleukin 14 alpha transgenic mouse (IL14αTG) that develops many of the features of SD in the same relative time frame as patients, hypergammaglobulinemia, autoantibodies, loss of salivary gland function, infiltration of salivary and lachrymal glands with lymphocytes, lymphocytic interstitial pneumonia, mild renal disease and eventually lymphoma [16–18]. Findings from this model have been corroborated in the NOD mouse, another model for SS, and in patients with SS and SD when possible [17,18]. The current studies investigate the expression of novel autoantibodies that were first identified in IL14αTG mice but subsequently shown to be present in NOD mice and in patients with SS and SD.
2. Materials and methods 2.1. Serum samples Sera were collected from mice using a protocol approved by the University at Buffalo IACUC committee (MED17105). Sera were collected from patients and normal controls by a protocol approved by University at Buffalo IRB committee (MED60510410E).
2.2. Determination of autoantibodies for SP-1, CA6, PSP, Ro and La Western blot were performed to evaluate the presence of autoantibodies for murine SP-1, CA6 and PSP following procedure described in previous studies [18]. ELISA's were performed to evaluate the presence of autoantibodies for murine Ro and La, human CA6 and PSP following procedure described in previous studies [16]. Antibodies to the human Ro and La were determined by ImmcoStripe Line Immunoassay for anti-nuclear antibody (Immco Diagnostics, Buffalo, NY, USA). The line Immunoblot assay consisted of immobilized auto-antigens (Ro52, Ro60 and La) and reacted as per the product protocol.
3. Results Previous studies recognized that the expression of Ro and La mRNA is increased in patients with SS and SD who have antibodies to Ro and La [19]. We observed that the mRNA for several salivary gland proteins, salivary protein 1 (SP-1), parotid secretory protein (PSP) and carbonic anhydrase 6 (CA6) was expressed at much higher levels in the mRNA of IL14αTG mice compared to control C57BL/6 mice. We therefore examined whether these proteins were target autoantigens in the SD of these mice. At the same time, we
L. Shen et al. examined whether the NOD mouse, which gets similar injury to the salivary and lachrymal glands as the IL14αTG mouse, produced autoantibodies to the antigens. Fig. 1A demonstrates that both IL14αTG mice as well as NOD mice produced autoantibodies recognizing SP-1, PSP and CA6. The control C57BL/6 mice did not make these autoantibodies. Both male and female IL14αTG mice produced these autoantibodies and mice that had antibodies to SP-1 generally also had antibodies to CA6 and PSP as well. Of 6 IL14αTG mice studied, 6 had antibodies to SP-1, 6 had antibodies to CA6 and 5 had antibodies to PSP. Similarly for 6 NOD mice studied, 6 had antibodies to SP-1, 6 had antibodies to CA6 and 4 had antibodies to PSP. Of 6 C57/BL6 control mice studied, none of them had antibodies to SP-1, CA6 or PSP. While antibodies to Ro and La are used as diagnostic criteria for SS and SD, we noted that only 25% of IL14αTG mice developed these autoantibodies. We therefore examined the time course of production of antibodies to Ro, La, SP-1 and CA6 in a cohort of IL14αTG mice. We have previously observed that IL14αTG mice have decreased salivary flow at 6 months of age with autoantibody deposition in the salivary glands. By 10 months of age IL14αTG mice have lymphocytic infiltration of the submandibular glands and by 14 months of age lymphocytic infiltration of the parotid glands as well [17]. As shown in Fig. 1B, antibodies to SP-1 and CA6 were present in more than 50% of IL14αTG mice by 6 months of age and present in all of the mice by 14 months of age. In contrast, none of the IL14αTG mice had antibodies to Ro or La at 6 months of age. By 14 months of age, 25% of IL14αTG mice had these autoantibodies. We next examined whether patients with SS and SD had antibodies to SP-1, CA6 and PSP. We evaluated autoantibodies to CA6 and PSP by ELISA assays using the human versions of these proteins. There is no human equivalent for SP-1, so we utilized a Western blot assay for the presence of this autoantibody using the murine protein. Fig. 2A demonstrates recognition of SP-1 by the sera of a patient with SD, but not the sera of a normal control. Next we evaluated the sera of 13 patients who met diagnostic criteria for SS and had disease for more than 5 years. Fig. 2B demonstrates that 62% of these patients had antibodies to Ro or La, 54% had antibodies to SP-1, 54% had antibodies to CA6 and 69% had antibodies to SP-1 or CA6. Only 20% of patients had antibodies to only CA6 or SP-1. In this cohort 18% had antibodies to PSP and 38% lacked antibodies to either Ro or La. All the patients had one of these autoantibodies. Evaluating a sex and age matched cohort of normal controls (Fig. 2C), 4.3% had antibodies to Ro or La, 4.3% had antibodies to SP-1 3% had antibodies to CA6 and 10% had antibodies to PSP. Using unpaired student's t test, the difference in Ro/La positivity (p = .0093) or SP-1/CA6 positivity (p = .0005) between SS patients and normal controls was highly significant. The difference in Ro/La positivity versus SP-1/CA6 positivity in SS patients was not significant (p = .400). Evaluating 10 patients with rheumatoid arthritis, but not secondary SS, none had antibodies to SP-1, while one had antibody to CA6, one to Ro, two to La and one to PSP (Fig. 2D). These patients were selected to be both RF positive (50%) and RF negative (50%). Because antibodies to SP-1 and CA6 occurred at an earlier time point in IL14αTG mice than antibodies to Ro and La, we also investigated a cohort of 29 patients who had idiopathic
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Figure 1 A. Evaluation of antibodies to SP-1, CA6 and PSP by Western blot in sera of IL14aTG mice, NOD mice and C57BL/6 mice; representative of 6 mice studied per group. B. Time course of antibodies to SP-1, CA6, Ro and La in the sera of 6 IL14αTG mice.
xerostomia and xerophthalmia for less than 2 years. These patients met at least 3 diagnostic criteria for SS and all the exclusion criteria. Fig. 2E demonstrates that of these patients, 31% had antibodies to Ro or La while 76% had antibodies to SP-1 or CA6. In this cohort, 45% had antibodies only to SP-1, 3% had antibodies to only CA6, no patients had antibodies only to Ro/La and 21% had none of these autoantibodies. In this cohort of patients, using unpaired student's t test, the difference in Ro/La positivity (p = .0187) or SP-1/CA6 positivity (p b .0001) between SS patients and normal controls was highly significant. The difference in Ro/La positivity versus SP-1/CA6 positivity in SS patients was also highly significant (p = .0004). Finally, we investigated 20 patients who met full diagnostic criteria for SS with positive salivary gland biopsies, but lacked antibodies to Ro or La. As shown in Fig. 2F, 45% of these patients had antibodies to SP-1 while 5% had antibodies to CA6.
4. Discussion The current studies demonstrate the presence of autoantibodies to SP-1, PSP and CA6 in two animal models of SS and in patients with SS. These autoantibodies occur earlier than antibodies to Ro and La and are present in patients who meet diagnostic criteria for SS but do not have antibodies to either Ro or La. SP-1 antibodies have the greatest specificity and
sensitivity for early SS. Autoantibodies to CA6 add additional sensitivity to the diagnosis of early SS along with SP-1, Ro and La. Carbonic anhydrase 6 is an enzyme involved with the buffering capacity of saliva that is found in both the cytoplasm and the secretory granules of the serous acinar cells in the submandibular and parotid glands [20]. Previous studies have demonstrated antibodies to CA6 in patients with SS, but the significance or timing of the production of these autoantibodies was not pursued [15]. In these studies we demonstrate that antibodies to CA6 occur very early in the course of SD. They occur rarely in normal controls or patients with rheumatoid arthritis lacking xerostomia and xerophthalmia. They rarely occur in patients who do not also have antibodies to SP-1. Parotid secretory protein is a secreted protein that is involved with binding and helping to clear various infectious agents [21,22]. It could easily become an autoantigen as it is bound to foreign proteins and cleaved by local proteases creating neoantigens. Interestingly mice and humans making antibodies to PSP generally make antibodies to CA6 at the same time. Antibodies to PSP occur only rarely in normal controls or patients with rheumatoid arthritis. Further study will be needed to determine whether testing for antibodies to PSP will increase the sensitivity of diagnosis for SS when antibodies to SP-1 and CA6 are already being done. Salivary protein 1 was identified in mice as an mRNA expressed at high levels in the submandibular glands and
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Figure 2 A. Western blot study showing antibodies to SP-1, CA6 and PSP in a representative patient with Sjogren's syndrome and normal control. This figure is representative of over 20 patients and normal controls studied in each group. B. Evaluations of antibodies to Ro, La, SP-1, CA6 and PSP in 13 patients with Sjogren's disease for more than 5 years. C. Evaluation of antibodies to SP-1, CA6, PSP, Ro and La in the sera of 23 normal controls. D. Evaluation of antibodies to SP-1, CA6, PSP, Ro and La in 10 patients with rheumatoid arthritis but not secondary Sjogren's syndrome. E. Evaluation of antibodies to SP-1, CA6, PSP, Ro and La in 29 patients with idiopathic xerostomia and xerophthalmia for less than 2 years. F. Evaluation of antibodies to CA6 and SP-1 in the sera of 20 patients with Sjogren's syndrome lacking antibodies to either Ro or La.
lachrymal glands of mice [23]. It was identified as one of the genes upregulated in the thymus by the autoimmune regulator gene (AIRE) to prevent development of T lymphocytes reactive to the salivary glands [24]. The function of the protein is unknown. The human homologue to SP-1 is also unknown. Interestingly there is a high degree of homology between SP-1 and a putative lipoprotein of Clostridium perfringens (gb EDT 72214.1). It is possible that reactivity to SP-1 occurs because of cross-reactivity to proteins produced by normal or abnormal flora in the respiratory and/or gastrointestinal tract. As a marker for SD, antibodies to SP-1 occur very early in the course of disease and occur in mouse models of SD and patients with SS and SD independent of antibodies to CA6, PSP, Ro or La. They have high sensitivity for SS and SD and are rarely found in patients with rheumatoid arthritis lacking secondary SS. Antibodies to SP-1 were found in 4.3% of normal controls, but it is unclear how many of these “normal controls” may have had early SS. The diagnostic criteria for SS and SD are undergoing continuous revision as more is learned about the pathophysiology of
these disorders. While Ro and La are autoantigens included in the diagnostic criteria for SS, many studies have identified patients with autoimmune SS and SD that do not have these autoantibodies [25–28]. Other autoantibodies such as the ones described in this manuscript may identify other subsets of patients and/or patients at different stages of the disease. One of the current challenges in SS and SD is to identify patients early enough so that therapy can result in recovery of their salivary and lachrymal glands. The autoantibodies to SP-1, CA6 and PSP may be helpful in this regard. New concepts regarding early events in SS and SD may provide therapeutic targets once these patients are identified [3,18].
Conflict of interest statement The authors declare that there are no conflicts of interest.
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