LAMP3 induces apoptosis and autoantigen release in Sjögren's syndrome patients

Tsutomu Tanaka, Blake M Warner, Toshio Odani, Youngmi Ji, Ying-Qian Mo, Hiroyuki Nakamura, Shyh-Ing Jang, Hongen Yin, Drew G Michael, Noriyuki Hirata, Futoshi Suizu, Satoko Ishigaki, Fabiola Reis Oliveira, Ana Carolina F Motta, Alfredo Ribeiro-Silva, Eduardo M Rocha, Tatsuya Atsumi, Masayuki Noguchi, John A Chiorini, Tsutomu Tanaka, Blake M Warner, Toshio Odani, Youngmi Ji, Ying-Qian Mo, Hiroyuki Nakamura, Shyh-Ing Jang, Hongen Yin, Drew G Michael, Noriyuki Hirata, Futoshi Suizu, Satoko Ishigaki, Fabiola Reis Oliveira, Ana Carolina F Motta, Alfredo Ribeiro-Silva, Eduardo M Rocha, Tatsuya Atsumi, Masayuki Noguchi, John A Chiorini

Abstract

Primary Sjögren's syndrome (pSS) is a complex autoimmune disease characterized by dysfunction of secretory epithelia with only palliative therapy. Patients present with a constellation of symptoms, and the diversity of symptomatic presentation has made it difficult to understand the underlying disease mechanisms. In this study, aggregation of unbiased transcriptome profiling data sets of minor salivary gland biopsies from controls and Sjögren's syndrome patients identified increased expression of lysosome-associated membrane protein 3 (LAMP3/CD208/DC-LAMP) in a subset of Sjögren's syndrome cases. Stratification of patients based on their clinical characteristics suggested an association between increased LAMP3 expression and the presence of serum autoantibodies including anti-Ro/SSA, anti-La/SSB, anti-nuclear antibodies. In vitro studies demonstrated that LAMP3 expression induces epithelial cell dysfunction leading to apoptosis. Interestingly, LAMP3 expression resulted in the accumulation and release of intracellular TRIM21 (one component of SSA), La (SSB), and α-fodrin protein, common autoantigens in Sjögren's syndrome, via extracellular vesicles in an apoptosis-independent mechanism. This study defines a clear role for LAMP3 in the initiation of apoptosis and an independent pathway for the extracellular release of known autoantigens leading to the formation of autoantibodies associated with this disease.ClinicalTrials.gov Identifier: NCT00001196, NCT00001390, NCT02327884.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
LAMP3 expression is increased in Sjögren’s syndrome patients, and increased expression is associated with serum autoantibodies. (A) LAMP3 mRNA expression levels in controls (healthy volunteers; open circle), non-SS (black dot) and primary SS (red dot) patients (Mean ± SD). (B) LAMP3 mRNA expression levels in controls, non-SS patients and SS patients, with or without presence of serum anti-SSA antibody (Mean ± SD). (C) Representative confocal immunofluorescent (IF) images (×40 magnification) of LAMP3 protein expression in (left image) a patient who does not meet SS criteria (non-SS), (middle image) a patient who meets criteria for primary SS (pSS) with median LAMP3 expression, (right image) and a patient (pSS#) with a characteristic lymphocytic focus adjacent to normal salivary gland epithelial tissue (ducts and acini). LAMP3 positive aggregates are present in acinar (open triangle) and ductal epithelia (arrow), the luminal debri (asterisks), and the lymphocytic foci (dashed outline). (D) IF intensity for LAMP3 protein expression in MSG biopsies from non-SS and pSS patients (Mean ± SD). (E) Differential expression of LAMP3 in human primary salivary gland epithelial cells (pSGECs) established from 7 SS patients and 8 healthy volunteers was analyzed by using RT-qPCR (open circle: SSA and SSB negative, closed circle: SSA and/or SSB positive). *P < 0.05, ANoVA; ††P < 0.01, †††P < 0.001, unpaired Student’s t-test.
Figure 2
Figure 2
LAMP3 overexpression increases TRIM21 expression and number and size of nuclear aggregates. Representative confocal immunofluorescent (IF) images (60X objective) of (A) TRIM21/SSA and (C) SSB protein expression in control HSG cells (empty) and LAMP3-overexpressing (OE) HSG cells. Nuclear aggregate size, number of nuclear aggregates per nuclei, and total IF intensity for (B) TRIM21/SSA and (D) SSB in control and LAMP3 OE cells. TRIM21/SSA aggregate size: N = 144 cells (control) and 558 cells (LAMP3-OE); TRIM21/SSA aggregate nember per nuclei: N = 149 cells (control) and 84 cells (LAMP3-OE); TRIM21/SSA total IF intensity: N = 177 cells (control) and 146 cells (LAMP3-OE). SSB aggregate size: N = 359 cells (control) and 430 cells (LAMP3-OE); SSB aggregate nember per nuclei: N = 130 cells (control) and 117 cells (LAMP3-OE); SSB total IF intensity:N = 146 cells (control) and 154 cells (LAMP3-OE). ***P < 0.001, ANoVA. Values shown are mean ± SD.
Figure 3
Figure 3
LAMP3 inhibits cell growth and induces caspase-dependent apoptosis. (A) HSG and (B) A253 cells were transfected with 1.5 μg pME18S-empty or pME18S-LAMP3 plasmid, and then 2 × 105 cells of each cells were re-plated 24 h post-transfection. The number of cells was counted 96 h after re-plating with Countess Automated Cell Counter (N = 3 independent experiment and 2 technical replicates of each experiment). (C,D) HSG cells and (E,F) A253 cells were transfected with different concentrations of pME18S-LAMP3 plasmid or a pME18S-empty plasmid as control. Seventy-two hours post-transfection, apoptotic cells were counted by using flow cytometry with APC Annexin V/7-AAD. Difference from control cells is shown (N = 4). (G,H) Number of apoptotic cells in control and LAMP3-overexpressing (OE) HSG cell cultures 24, 48 and 72 h after transfection, assessed by flow cytometry using APC Annexin V/7-AAD. Difference from control cells is shown (N = 3). (I,J) Number of apoptotic cells in GFP+ control, LAMP3-OE and LAMP1-OE HSG cell cultures 48 h after transfection, assessed by flow cytometry using APC Annexin V/7-AAD. Difference from control cells is shown (N = 4). (K,L) Number of apoptotic cells in GFP+ control, LAMP3-OE and LAMP1-OE A253 cell cultures 48 h after transfection, assessed by flow cytometry using APC Annexin V/7-AAD. Difference from control cells is shown (N = 4). (M,N) Control and LAMP3-OE HSG cells were incubated with or without 20 μM Z-VAD-FMK (Z-VAD) for 20 h. Extent of apoptosis was determined by flow cytometry using APC Annexin V/7-AAD. Difference in rate of Annexin V+ cells in LAMP3-OE cell culture treated with or without 20 μM Z-VAD from that in control cell culture is shown (N = 4). *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t-test. Values shown are mean ± SD.
Figure 3
Figure 3
LAMP3 inhibits cell growth and induces caspase-dependent apoptosis. (A) HSG and (B) A253 cells were transfected with 1.5 μg pME18S-empty or pME18S-LAMP3 plasmid, and then 2 × 105 cells of each cells were re-plated 24 h post-transfection. The number of cells was counted 96 h after re-plating with Countess Automated Cell Counter (N = 3 independent experiment and 2 technical replicates of each experiment). (C,D) HSG cells and (E,F) A253 cells were transfected with different concentrations of pME18S-LAMP3 plasmid or a pME18S-empty plasmid as control. Seventy-two hours post-transfection, apoptotic cells were counted by using flow cytometry with APC Annexin V/7-AAD. Difference from control cells is shown (N = 4). (G,H) Number of apoptotic cells in control and LAMP3-overexpressing (OE) HSG cell cultures 24, 48 and 72 h after transfection, assessed by flow cytometry using APC Annexin V/7-AAD. Difference from control cells is shown (N = 3). (I,J) Number of apoptotic cells in GFP+ control, LAMP3-OE and LAMP1-OE HSG cell cultures 48 h after transfection, assessed by flow cytometry using APC Annexin V/7-AAD. Difference from control cells is shown (N = 4). (K,L) Number of apoptotic cells in GFP+ control, LAMP3-OE and LAMP1-OE A253 cell cultures 48 h after transfection, assessed by flow cytometry using APC Annexin V/7-AAD. Difference from control cells is shown (N = 4). (M,N) Control and LAMP3-OE HSG cells were incubated with or without 20 μM Z-VAD-FMK (Z-VAD) for 20 h. Extent of apoptosis was determined by flow cytometry using APC Annexin V/7-AAD. Difference in rate of Annexin V+ cells in LAMP3-OE cell culture treated with or without 20 μM Z-VAD from that in control cell culture is shown (N = 4). *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t-test. Values shown are mean ± SD.
Figure 4
Figure 4
LAMP3-induced activation of caspase-3 is independent of endoplasmic reticulum stress. Caspase-3 (CASP3) mRNA expression levels were measured by qRT-PCR in control and LAMP3-overexpressing (OE) (A) HSG and (D) A253 cells. Western blot analysis was performed to measure (B,E) caspase-3 protein, (C,F) ATF4, spliced XBP1 (XBP1s) and unspliced XBP1 (XBP1u) protein levels in HSG and A253 control and LAMP3-OE cells, respectively. All ratios are relative value compared with control. Uncropped images are provided in Supplementary Figure S4.
Figure 5
Figure 5
LAMP3-induced accumulation of autoantigens and release via extracellular vesicles. (A) Images were collected by immunofluorescent microscopy at 100X magnification. LAMP3-overexpressing (OE) cells show a membranous and vesicular pattern of LAMP3 expression. Some of the vesicles appear just below or budding from the plasma membrane and colocalize with TRIM21/SSA and SSB (open triangles). TRIM21/SSA, SSB, α-fodrin and cleaved (cl.) α-fodrin protein levels in control and LAMP3-OE cells. Rows 2 and 4 are further enlarged images of specific cells shown in row 1 and 3. The specific cells in the merged image are boxed for clarity. Indicated protein expression in (B) HSG and (C) A253 cells, as determined by Western blotting. (D) Protein concentration in mixture of extracellular vesicles (EVs) isolated from control and LAMP3-OE HSG cells. Data are presented as relative change in expression compared with control. Western blotting analysis of TRIM21/SSA and SSB in EVs isolated from control and LAMP3-OE (E) HSG and (F) A253 cells. (G) Western blotting of α-fodrin and cl. α-fodrin in EVs from control and LAMP3-OE HSG cells. Protein levels were normalized to α-tubulin or flotillin-1 level. Uncropped images are provided in Supplementary Figure S5. ***p < 0.001, unpaired Student’s t-test.
Figure 5
Figure 5
LAMP3-induced accumulation of autoantigens and release via extracellular vesicles. (A) Images were collected by immunofluorescent microscopy at 100X magnification. LAMP3-overexpressing (OE) cells show a membranous and vesicular pattern of LAMP3 expression. Some of the vesicles appear just below or budding from the plasma membrane and colocalize with TRIM21/SSA and SSB (open triangles). TRIM21/SSA, SSB, α-fodrin and cleaved (cl.) α-fodrin protein levels in control and LAMP3-OE cells. Rows 2 and 4 are further enlarged images of specific cells shown in row 1 and 3. The specific cells in the merged image are boxed for clarity. Indicated protein expression in (B) HSG and (C) A253 cells, as determined by Western blotting. (D) Protein concentration in mixture of extracellular vesicles (EVs) isolated from control and LAMP3-OE HSG cells. Data are presented as relative change in expression compared with control. Western blotting analysis of TRIM21/SSA and SSB in EVs isolated from control and LAMP3-OE (E) HSG and (F) A253 cells. (G) Western blotting of α-fodrin and cl. α-fodrin in EVs from control and LAMP3-OE HSG cells. Protein levels were normalized to α-tubulin or flotillin-1 level. Uncropped images are provided in Supplementary Figure S5. ***p < 0.001, unpaired Student’s t-test.
Figure 6
Figure 6
LAMP3-induced autoantigens release was independent of apoptosis. (A) Protein concentration in mixture of extracellular vesicles (EVs) isolated from control and LAMP3-OE HSG cells treated with or without Z-VAD. Data are presented as relative change in expression compared with control cells. (B) Western blotting analysis of TRIM21/SSA, SSB and α-fodrin in EVs isolated from HSG control and LAMP3-OE cells treated with or withour Z-VAD. Protein levels of (C) TRIM21/SSA, (D) SSB and (E) α-fodrin were normalized to flotillin-1 level, and the value was plotted. Uncropped images are provided in Supplementary Figure S6. **p < 0.01, ***p < 0.001, unpaired Student’s t-test.

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