atlas of antinuclear antibodies · atlas of antinuclear antibodies. the autoantibody testing by...

Atlas of Antinuclear Antibodies

The autoantibody testing by indirect immunofluorescence (IIF) has fulfilled the very important roles for the diagnosis and treatment of the patients with autoimmune disorders since its first report by Friou in 1957.The technique has many advantages for detecting and screening both nuclear and cytoplasmic antibodies in general and categorizing them by their staining patterns. Since the recognized antigens of the ANA (antinuclear antibodies) comprise many nuclear components or proteins and sometimes it is difficult to distinguish them by their staining patterns, these studies have contributed much to the elucidation and understanding of the autoantibodies in connective tissue diseases.

* Photos of cell staining in this photo book were taken by using FLUORO HEPANA TEST (code No. 4210, 4220). Specific staining parts were detected as fluorescent staining (green) and non specific staining parts would be appeared as red because of Evans Blue pigment. Depends on cases, when both parts were over lapped, it would look yellow.

　　　PCNA

　　　Ribosome

　　　Lysosome

　　　Golgi complex　　Mitochondria


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Structure and function of eukaryotic cell and outlines of cell cycle

One of the major advantages of detecting and screening autoantibodies by IIF is to find out the localization of recognized cellular antigens in various cell cycles by their staining patterns. It becomes very important to realize the structure of the cell related to the function and localization of the antigens in the cell.

■ Structure and function of cellGeneral structure of the eukaryotic cell as HEp-2 cell is shown in Fig. 1. Inside the cell, various organelles including the nucleus, endoplasmic reticulum, golgi apparatus, mitochondria, peroxisomes and lysosomes are recognized. The cell surface is covered by plasma membrane, and the space between the plasma membrane and organelles are occupied by insoluble cytoskelton and soluble cytoplasm.

Nucleus

The nucleus is covered by bi-layer nuclear membrane. The outer nuclear membrane is connected with endoplasmic reticulum and transport of various substances between the nucleus and cytoplasm occurs through nuclear pore complexes. The inner nuclear membrane is connected with lamina. In interphase cells, chromosomes are dispersed into chromatins throughout the nucleoplasm. The chromatins are composed of DNA, histones, and other non-histone proteins. In the granular components of nucleus, precursor mRNA (heterogeneous nuclear RNA; hnRNA) and small RNA-protein complexes (small nuclear ribonucleoproteins; snRNP) for splicing of hnRNA can be found.In the nucleolus, ribosomal RNA precursors are transcribed and after their processing and assembly, ribosomes are transported to the cytoplasm.

Cytoplasm

Endoplasmic reticulum (ER)ER are divided into rough surface RER which binds ribosomes on its surface and smooth surface SER. RER is the main site of protein synthesis in the cell and the SER is found abundantly in cells like Leydig cells in the testis which synthesize and secrete steroid hormones. Oxidoreductase systems of NADH and NADPH in the SER membrane are concerned with unsaturation and ω-oxidation of fatty acids and detoxication of xenobiotic substances.

Golgi apparatusGolgi apparatus is composed of stacks of cisterna and divided into cis, medial and trans regions. It is mainly concerned with the modification (addition of sugar chains) and sorting of the synthesized proteins in RER for secretion and transport in cells.

MitochondriaMitochondria are oval in structure. Respiratory chain and ATP synthetase complex for ATP production are in its inner membrane. Enzymes for TCA cycle, fatty acid oxidation and amino acid metabolism, and own protein synthesis system with mitochondrial DNA are in its matrix.

LysosomeLysosome is an organelle with monolayer membrane, and it is mainly concerned with the hydrolysis of waste products and toxic substances (proteins, mucopolysaccharides, glycolipids and nucleic acids) by their respective enzymes localized in the matrix where low pH is maintained by the action of H+-ATPase.

centriole

endoplasmic reticulum

cytoskeleton

cell membrane

cytoplasm

nucleolus

nucleus

ribosome

nuclear membrane

mitochondria

Golgi apparatus

lysosome

Fig. 1 HEp-2 cell



Table 1 Antigens recognized by autoantibodies in connective tissue diseases

antinuclear antibodies localization recognized antigens relevant diseases

anti-ssDNA antibodyanti-dsDNA antibodyanti-polyADP ribose antibodyanti-Scl-70 antibodyanti-centromere antibodyanti-histone antibody

chromatins single stranded DNA (ssDNA)double-stranded DNA (dsDNA)poly ADP riboseDNA topoisomerase Icentromerehistone

SLE etc.SLESLESScCRESTDrug induced lupus

anti-U1RNP antibodyanti-Sm antibodyanti-U2RNP antibodyanti-SS-A/Ro antibodyanti-SS-B/La antibodyanti-PCNA antibodyanti-Ku antibody

nucleus U1RNPU1, U2, U4/U6, U5 RNPU2RNPhY1-hY5 RNPRNA polymerase III transcriptionDNA polymerase δ auxiliary factorDNA-dependent protein kinase activation factor

MCTDSLEOverlap syndromeSS etc.SSSLEOverlap syndrome

anti-U3RNP antibodyanti-7-2(Th)RNP antibodyanti-RNA polymerase I antibodyanti-PM-Scl antibodyanti-NOR-90 antibody

nucleolus U3RNP (Fibrillarin)RNaseP, RNaseMRPRNA polymerase I11 proteins complexproteins complex (20-110 kD)nucleolus NOR (hUBF)*

SScSScSScOverlap syndromeSSc

anticytoplasmic antibodies localization recognized antigens relevant diseases

anti-Jo-1 antibodyanti-ribosomal antibodyanti-mitochondrial antibodyanti-SS-A/Ro antibodyothers (anti actin antibody etc.)

cytoplasm histidyl-tRNA synthetase60S ribosomal subunitmitochondriahY1-hY5 RNP

PM/DMSLEPBCSS etc.

*human Upstream Binding Factor

SLE: systemic lupus erythematosus, SSc: systemic sclerosis, MCTD: mixed connective tissue disease, SS: Sjögren's syndrome, PM/DM: polymyositis/dermatomyositis, RA: rheumatoid arthritis, PBC: primary biliary cirrhosis, AIH: autoimmune hepatitis

Cell cycle

The cycle of the cell can be divided into interphase and mitosis (M phase). The interphase is subdivided into 3 successive periods: G1(Gap 1), S(Synthesis), and G2(Gap 2) phases. The dispersed chromatin network of the nucleoplasm in the interphase begins to condense in M phase, assembling into pairs of chromatids linked to each other at their centromeres. Mitosis is divided into prophase, prometaphase, metaphase, anaphase and telophase.

*Staining patterns of anti-centromere antibody in photo.



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Fluorescence patterns of autoantibodies


■ Typical patterns of ANA using HEp-2 cellsRodent tissue sections which were used as substrates during early periods of ANA testing have been replaced by HEp-2 cells (immortalized cells orginated from human laryngeal carcinoma) grown as a monolayer on the slide. The HEp-2 cells have much larger structures and higher cell division rates, so the detailed nuclear and cytoplasmic staining patterns can be recognized in the cell populations of different cell cycles. It is noted that HEp-2 cells exhibit more than 30 different patterns, some are antigen-specific but some are similar with different specificities, and the mixed patterns by different autoantibodies in patients' sera are frequently observed. etailed nuclear and cytoplasmic staining patterns can be recognized in the cell populations of different cell cycles. It is noted that HEp-2 cells exhibit more than 30 different patterns, some are antigen-specific but some are similar with different specificities, and the mixed patterns by different autoantibodies in patients' sera are frequently observed.

Negative

No significant nuclear and cytoplasmic staining. Depending on the sensitivity of the fluorescence microscope, faint fluorescence can be observed, which is considered negative.

In case of positive, staining patterns are interpreted.

Homogeneous nuclear staining

Uniform diffuse fluorescence staining of the entire nucleus in interphase cells. The chromosomal regions in mitotic cells give intense fluorescence.

photo 1

photo 2HEPASERA-I**

** HEPASERA-1 is MBL's ANA reference sera (Homogeneous pattern, Speckled pattern, Nucleolar pattern, Discrete speckled pattern).




Peripheral nuclear staining

Uniform diffuse nuclear staining similar to homogeneous staining, with greater intensity at its outer rim. The chromosomal region in mitotic cells has uniform staining with greater intensity at its outer rim. Most of the sera showing this pattern give homogeneous staining when sera are diluted.

Speckled nuclear staining

Fine or coarse speckles are distributed in the entire nucleus of interphase cells. The chromosomal region shows no flurorescence in most of the mitotic cells.

Centromere (Discrete speckled) staining

40 to 80 discrete speckles are stained in the nucleus of interphase cells. Speckles aligned in the chromosomal region of mitotic cells can be seen.

photo 4HEPASERA-I**

photo 5HEPASERA-I**

photo 3




Nucleolar staining

Homogeneous or speckled (clumpy) staining of the nucleolus of interphase cells are seen by various nucleolar autoantibodies.

Other staining patterns and antigens,

Cell cycle-dependent pleomorphic staining

proliferating cell nuclear antigen (PCNA)

Nuclear dots staining minute nuclear antigens; p80 coilin, Sp100 etc.

Nuclear membranous staining lamin, nuclear pore complex

Cytoplasmic staining cytoplasmic antigens; enzymes such as aminoacyl-tRNA synthetases, organelles such as mitochondria and ribosome, cytoskelton, mitotic spindle apparatus

photo 6HEPASERA-I**

** HEPASERA-1 is MBL's ANA reference sera (Homogeneous pattern, Speckled pattern, Nucleolar pattern, Discrete speckled pattern).



Mixed patterns

■ Mixed patternsMixed staining patterns are observed in a patient's serum containing more than one autoantibody. Since it is sometimes difficult to distinguish different autoantibodies, the patterns can often be confirmed by titrating out the serum for its quantitative evaluation. It is also useful to observe the staining of the chromosomal region and surrounding cytoplasm in mitotic cells. The following photos show examples of mixed patterns.

indicate staining of the homogeneous, speckled and the mixed patterns respectively

The serum sample with the speckled pattern (photo 7) was mixed with another serum with homogeneous pattern (photo 8) at 1:1 ratio. The mixed sample shows the speckled pattern in interphase cells combined with chromosomal staining of mitotic cells. (photo 9) The staining pattern is suggestive of coexistence of the speckled and the homogeneous patterns.

photo 7Speckled nuclear staining

photo 9Speckled nuclear staining and Homogeneous nuclear staining mixed

photo 8Homogeneous nuclear staining



Mixed patterns

show another example of the mixed pattern

A serum sample with the anti-centromere antibody (photo 10) and the anti-mitochondrial antibody (photo 11) gives characteristic staining of nucleus and cytoplasm suggestive of the coexistence of both antibodies (photo 12).

photo 12The characteristic staining of nucleus and cytoplasm suggestive of the coexistence of both antibodies

photo 11Anti-mitochondrial antibody

photo 10Anti-centromere antibody



Autoantibodies recognizing chromatin related antigens

Fluorescence patterns of nuclear autoantigens

■ Autoantibodies recognizing chromatin related antigens

Chromatins are composed of dsDNA and its binding proteins (histones and non-histone proteins). 146 base pairs of dsDNA wind around histone core (histone octamer composed of 2 molecules each of H2A, H2B, H3 and H4 subunits) to make a nucleosomal unit which connect to each other through a DNA linker with histone H1 subunit. In interphase cells, chromatins are dispersed in the nucleoplasm and condensed to form chromatids (chromosomes) in mitotic cells.

Anti-dsDNA antibody

nucleosome, the fundamental structural unit of chromatin,consists of DNA wound around a core particle of histone proteins

octameric histone core(two each of H2A,H2B,H3,H4 proteins)

chromatin fiber

chromosome "beads-on-a-string" form of chromatin

DNA

DNA

photo13Peripheral staining

photo14 　　AF/CDC-1*Homogeneous staining




Pattern Peripheral and homogeneous staining in interphase cells with strong chromosomal staining in mitotic cells. At lower serum dilutions (x 20-x 40), the antibodies tend to give peripheral patterns and often show homogeneous staining at higher serum dilutions. Peripheral patterns are more frequently found when rodent tissue sections are used as substrates.

Antigen Double stranded DNA(dsDNA). Anti-ssDNA antibodies do not give fluorescence.

Other analytical method

IIF, ELISA, RIA, CLEIA

Clinicalsignificance

The antibody is found flequently and specifically in SLE patients with active renal disease. The presence of Anti-dsDNA is a major criteria for the classification of SLE.

References 5, 6

Anti-DNA histone complex antibody (anti-DNP antibody / anti-nucleosomal antibody)

Pattern Homogeneous nuc lear s ta in ing in interphase cells and intense fluorescence in the chromosomal region in mitotic cells.

Antigen Histone-DNA complex with the antigenic epitope in DNA-histone binding sites.

Otheranalytical method

ELISA

Clinical significance

SLE, lupus nephritis

References 7, 8

* Reference serum from Centers for Disease ControlSLE: systemic lupus erythematosus, SSc: systemic sclerosis, MCTD: mixed connective tissue disease, SS: Sjögren's syndrome, PM/DM: polymyositis/dermatomyositis, RA: rheumatoid arthritis, PBC: primary biliary cirrhosis, AIH: autoimmune hepatitis

photo15 kinetoplast of Clithidia luciliae is stained.

photo16




Anti-histone antibody

Pattern Similar to the former antibody

Antigen Histone H1, H2A, H2B, H3, H4, H2A-H2B complex (major antigen in IIF method) and H3-H4 complex.


ELISA, WB


SLE (antibodies to H1 and H2A), drug-induced lupus (antibodies to H2A-H2B complex)

References 9

Anti-Ku antibody

Pattern Speckled (patchy) nuclear staining (sometimes described as homogeneous staining) with no chromosomal staining in mitotic cells.Monoclonal antibodies to the 70kD subunit give similar patterns.

Antigen DNA binding 80kD/70kD heterodimer protein (DNA-dependent protein kinase activator). Antibodies targeting either subunit or the complex are reported.


DID, Immunoprecipitation


SLE, SSc (USA) / polymyositis/scleroderma overlap syndrome (Japan)

References 1, 4, 10, 11

Anti-Scl-70 antibody (anti-Topoisomerase I antibody)

Pattern Homogeneous (dense fine speckled to homogeneous) staining with chromosomal staining in interphase cells.Nucleolar staining is often observed.

Antigen DNA Topoisomerase I


DID, ELISA, WB, CLEIA


Antibodies to topoisomerase I are generally found in systemic sclerosis with high sensitivity and their presence constitutes one of the diagnostic criteria of the disease.

References 12

photo17

photo 18AF/CDC-9*




Anti-centromere antibody

Pattern Centromere or discrete speckled staining. 40-80 fine speckles are stained in the nucleus of interphase cells. The speckles are characteristically aligned at the chromosomal region in mitotic cells. Paired centromeric fluorescence staining is observed in the chromosomal spread preparations.

Antigen Centromere proteins; CENP-B (80kD), CENP-A (17kD), CENP-C (140kD)


ELISA, WB, CLEIA


These antibodies are consistently found in patients with the limited form of SSc and their presence constitutes a diagnostic criteria of SSc. These antibodies are also found in PBC.

References 13, 14

centromereheterochromatinCENP-B 80kD

inner plateCENP-C 140kD

middle zone3F3/2 antigen

outer plateCENP-F

fibrous coronaCENP-E,dynein

kinetochore

pairing domain

The constricted region where two sister chromatids join along is the centromere. CENP is a specific protein in this region. Microtubules of the spindle apparatus attach to kinetochore when the chromatid separation occurs.


photo 19AF/CDC-8*

photo 202 fluorescent dots are visible for each chromosomal pair. (hypotonic spread preparation of mitotic chromosomes)



Autoantibodies recognizing non-chromatin related proteins

■ Autoantibodies recognizing non-chromatin related proteins

The genetic information of DNA is transcribed to precursor mRNA (heterogeneous nuclear RNA, hnRNA). The small nuclear RNAs (small nuclear RNA, snRNA) such as U1, U2, U4/U6 and U5 RNA are involved in the splicing of hnRNA. These RNAs are complexed with proteins to form ribonucleoproteins (RNPs).

Anti-U1RNP antibody

Pattern Coarse speckled (larger granular) nuclear pattern with no nucleolar and cytoplasmic staining. The chromosomal regions do not stain in mitotic cells.

Antigen 3 kinds of polypeptides bound to U1 RNA; U1RNP-68kD, U1RNP-A(34kD), U1RNP-C(23kD)


DID, ELISA, WB, Immunoprecipitation, CLEIA

Clinical ignificance

The presence of the antibodies to U1RNP constitute one of major diagnostic criteria for MCTD.

References 15, 16

DNA

RNA polymerase II

mRNA

precursor mRNA(hnRNA) introns introns

splicing

splicing

U1RNP

U2RNP

U5RNPU4/U6RNP

RNP

SmA

CBB'DEFG

U1 RNA

photo21




Anti-Sm antibody

Pattern Coarse speckled staining similar to those of anti-U1RNP antibody.

Antigen U1, U2, U4/U6 and U5 RNA-bound proteins with 5 kinds of epitopes, B'/B(29kD/28kD), D(16kD), E(12kD), F(11kD) and G(10kD). The main epitopes comprise B'/B and D polypeptides. Antibodies to E-F-G complex as dominant epitopes are also reported.




Marker antibody for SLE. The presence of anti-Sm antibody is included in the criteria for the classification of SLE by the American College of Rheumatology.

References 17

Anti-U2RNP antibody / Anti-U1U2RNP antibody

Pattern Coarse speckled nuclear staining similar to those of anti-U1RNP antibody.

Antigen U2RNA-bound proteins with 2 kinds of epitopes on A'(32kD) and B"(28.5kD).


Immunoprecipitation


Polymyositis/scleroderma overlap syndrome

References 3, 15, 16

photo 22AF/CDC-5*

photo 23





Anti-hnRNP antibody

Pattern Large speckled (granular) nucleoplasmic staining with no chromosomal fluorescence in mitotic cells. The antibodies frequently coexist with anti-U1RNP and anti-Sm antibodies, and give coarse speckled patterns.

Antigen hnRNA bound proteins-A/B, A1, A2, C, I


ELISA, WB


These antibodies were first reported in patients with MCTD. Antibodies to the A/B polypeptides and those to the A1 and A2 polypeptides occur in SLE and rheumatoid arthrit is, respectively. Recently, anti-hnRNP-I antibodies with weak nuclear staining specific for scleroderma and anti-hnRNP-C antibodies with speckled nuclear staining in scleroderma were reported.

References 18, 19, 20, 21

Anti-SS-A/Ro antibody

Pattern Fine speckled nuclear staining observed in acetone-fixed slides, but not in alcohol-fixed ones. The chromosomal region shows no fluorescence in mitotic cells.

Antigen Y1-Y5RNA-bound proteins (52kD, 60kD)

Other analytical method DID, ELISA, WB, Immunoprecipitation, CLEIA

Clinical significance These antibodies occur most frequently in patients with SS, and those with SLE. Sometimes found in patients with rheumatoid arthritis, myositis or scleroderma. The presence of the antibodies in the mother is associated with neonatal lupus and congenital heart block. The presence of anti-SS-A antibodies constitutes one of the diagnostic criterion of SS in Europe.

References 22

SS-B/LaRo 60 kD

Ro 52 kDLa

Y-RNA

SS-A/Ro

photo 24

photo 2　　　　　　　　　　　　　　　　　　　F/CDC-7*




Anti-SS-B/La antibody

Pattern Fine dense speckled nuclear staining is observed in interphase cells with no chromosomal fluorescence in mitotic cells.

Antigen 48 kD SS-B/La protein, a termination factor for RNA polymerase III transcription




The antibodies are found in patients with SS and SLE. As with anti-SS-A antibodies, the presence of these antibodies is associated with neonatal lupus and congenital heart block. The antibodies were reported to be frequently positive in patients with recurrent annular erythema. The presence of anti-SS-B antibodies constitutes one of the diagnostic criterion of SS in Europe.

References 23

Anti-p80 coilin antibody

Pattern Granular or few nuclear dots staining**, 0 to 6 dots (ave. 2 dots) per nucleus fluorescence. The distinct large dotty fluorescence can be seen in cells during S and G2 phases, and mitotic cells show no staining.

Antigen p80 coilin, the 80kD protein composing the coiled body in nucleoplasm.


WB


These antibodies occur in some patients with SS and those with primary biliary cirrhosis.

References 24, 25

photo 26AF/CDC-2*

photo 27





Anti-Sp100 antibodies

Pattern Granular or multiple (1 to 24) nuclear dots staining**.

Antigen Sp100 protein. Mult iple nuclear dots staining is also observed by the antibodies to promyelocytic leukemia (PML) protein, NDP53 (a binding protein to PML oncogenic domain including Sp100).


WB


These antibodies occur in some patients with PBC.


* Reference serum from Centers for Disease Control** Since the numbers of staining dots seen in few nuclear dots and multiple nuclear dots patterns overlap each other, it is sometimes difficult to distinguish these patterns. These patterns are often called granular or nuclear dots patterns.

photo 28



Autoantibodies recognizing nucleolus related proteins

■ Autoantibodies recognizing nucleolus related proteins

Fibrillar component:

Fibrillar center:

Granular component:Maturation of the rRNA is carried out and transported outside of the nucleolus.antigen: PM-Scl, B23, NO55

The processing of pre-rRNA is performed.antigen: fibrillarin

Nucleolus organizing regions (NOR) are the main s i tes of nucleol i format ion af ter cel l division. The precursor rRNA is transcribed from rRNA gene by RNA polymerase I.antigen: RNA-polymerase I ~ III, NOR-90

mRNA

cytoplasm

nucleus

endoplasmic reticulum

nuclear membrane

ribosome

ribonucleoprotein particle

precursor rRNA

chromatin includingrRNA gene

nucleolus

Nucleolus

The nucleolus is composed of the central fibrillar region and the outer granular component. In the fibrillar center, nucleolus organizer region (NOR) is related to the reformation of the nucleolus after cell divison.

Anti-U3RNP antibody (Anti-fibrillarin antibody)

Pattern Clumpy nucleolar staining in interphase cells with chromosomal region fluorescence of mitotic cells.

Antigen U3RNA-bound protein; fibrillarin (34kD)


Immunoprecipitation


These antibodies occur in SSc (mainly observed in patients with diffuse cutaneous scleroderma not associated with arthritis and lung fibrosis), and in hepatocellular carcinoma.

References 29, 30

photo 29





Anti-RNA polymerase I antibody

Pattern Punctate or speckled nucleolar staining in interphase cells. The chromosomal regions show no staining in mitotic cells.

Antigen RNA polymerase I, II and III component proteins (more than 10 proteins ranged from 12.5 to 210kD). The autoantibodies targeting RNA polymerase II or III, and those reacting to more than one RNA polymerase were reported.


Immunoprecipitation


These antibodies occur in patients with SSc, frequently associated with patients with severe internal organ involvement (mainly lung and kidney) and poor prognosis.


Anti-7-2RNP antibody (Anti-Th antibody)

Pattern Dotty nucleolar staining

Antigen RNaseP(8-2RNP), RNaseMRP(7-2RNP)


Immunoprecipitation


These antibodies occur in patients with scleroderma, mainly associated with limited cutaneous involvement.

References 29

Anti-PM-Scl antibody

Pattern Homogeneous or diffuse nucleolar staining with no chromosomal staining in mitotic cells.

Antigen The protein complex of more than 10 nucleolar proteins including 75kD and 100kD polypeptides.


These antibodies occur in patients with polymyositis/scleroderma overlap syndrome in Europe and America, but not in Japan.

References 29

photo 30

photo 31

photo 32




Anti-NOR-90 antibody

Pattern Coarse speckled or granular nucleolar staining in interphase cel ls. Several intensely stained dots can be seen in the chromosomal regions in mitotic cells.

Antigen 90kD/92kD proteins in nucleolus organizer region, recognizing RNA polymerase I transcription factor, hUBF (human upstream binding factor) in the fibrillar center.


DID, ELISA, WB, Immunoprecipitation


These antibodies occur in patients with SSc frequently associated with Raynaud's phenomenon, also those with hepatocellular carcinoma and SS in Japan.

References 30, 31

Anti-RNA helicase antibody

Pattern Dense speckled nucleolar staining with no chromosomal staining in mitotic cells.

Antigen 100 kD nucleolar RNA helicase protein


Immunoprecipitation


These antibodies occur in patients with SSc, SLE or watermelon stomach disease.

References 32, 33

Other autoantibodies to nucleolar antigens:Anti-B23/nucleophosmin antibodies occur in patients with hepatocellular carcinoma and show homogeneous nucleolar staining. [References: 30, 34]Anti-No55 antibodies occur in patients with interstitial cystitis and show homogeneous nucleolar pattern. [Reference: 35]

photo 33

photo 34




Autoantibodies recognizing cell cycle related antigens

■ Autoantibodies recognizing cell cycle related antigensThe remarkable differences in the patterns and intensities of nuclear staining among HEp-2 cells on the slide indicate the presence of autoantibodies targeting the antigens which show changes in their quantities, localizations and molecular structures dependent on the cell cycle. Sometimes, coexistence of more than two autoantibodies is suggested by the pleomorphic staining patterns. However the details are not clear.

Anti-PCNA antibody

PCNA: Proliferating Cell Nuclear Antigen

Pattern Most intense nuclear staining occur in S phase cells. The antibodies are characterized by the variation of staining patterns in the cells in different cell cycles; sparse fine speckled nuclear staining in late G1 phase cells, dense homogeneous staining in S phase cells, and coarse nuclear staining with no nucleolar decoration in late S phase cells. The chromosomal regions show no staining in mitotic cells. When the antibodies coexist with other antibodies, it is sometimes useful to confirm the specific staining by serial serum dilutions.

Antigen 34kD auxiliary protein of DNA polymerase δ. Recently, it was reported that PCNA antigen forms a macromolecule complex with Ki antigen.


DID, WB


These antibodies occur in patients with SLE (1-2%), but rarely seen in other diseases.


photo 35




Anti-Na antibody

Pattern Nuclear speckled staining is seen mainly in G2 phase cells, but cell cycle-dependent polymorphic staining is not remarkable compared with that of anti-PCNA antibody. The chromosomal regions of mitotic cells show speckled staining similar to those given by anti-centromere antibody. The cleavage furrow observed in the telophase of mitosis and the midbody after cell division is stained by the antibodies.

Antigen Myosin related antigen


The presence of this antibodies was reported in patients with SLE.

References 39, 40

Anti-CENP-F antibody was reported to give similar staining patterns to anti-Na antibody. They occur in malignant thoracic tumors such as lung cancer and recognize one of the centromere proteins (CENP-F 367kD). [References:44, 73]Anti-midbody (MSA-2) antibody occuring in scleroderma show negative or very weak nuclear staining in interphase cells compared with anti-Na antibody. [References: 43, 44]Anti-MSA-3 antibody shows speckled staining in some of the interphase cells, but do not stain the cleavage furrow in telophase cells. The antibodies were reported to occur in association with cancer of the respiratory system. [Reference: 44]It was also reported that anti-DNA topoisomerase II antibodies are observed in hepatocellular carcinoma. [Reference: 30]

photo 36 photo 37





staining patterns of the antibodies recognizing cell cycle related antigens

The photos shown below are staining patterns of the antibodies recognizing cell cycle related antigens, observed in MBL Co., Ltd.. Details of the antigens and the clinical significance are not determined.

Many proteins have been found to be associated with proliferation of the cells. Among them are some of the antinuclear antibodies with cell cycle dependency. The autoantibodies observed in patient sera may provide useful tools for studying the structure and function of these autoantigens. [Reference: 45]

photo 38Homogeneous nuclear staining in some of the interphase cells with more intensive staining of chromosomal region in mitotic cells.

photo 40Some of the nucleoli are stained.

photo 39Weak nuclear f luorescence in some of the cells with cytoplasmic staining.

photo 41No nuclear staining in interphase cel ls with intense chromosomal staining in mitotic cells.



Autoantibodies recognizing nuclear membrane related antigens

■ Autoantibodies recognizing nuclear membrane related antigens

nucleus

cytoplasm

outer membraneinner membrane

ribosome

endoplasmic reticulumpore membrane

nuclear laminaLAP1 LAP2

lamin Alamin Blamin C

nuclear membranegp210

Nuclear membrane is a lipid bi-layer, and its inner membrane is connected with nuclear lamina. Ribosomes and endoplasmic reticulum are bound to the outer membrane. The nuclear membrane is perforated by multiple nuclear pores through which transportation of molecules between the nucleus and the cytoplasm occurs.

Anti-lamin antibody

Pattern L inear f i ne s ta in ing o f the nuc lea r membrane and homogeneous nuclear staining associated with nuclear r im staining are observed. The staining is similar to peripheral pattern. However, the chromosomal region show no staining in mitotic cells.

Antigen Nuclear lamins (intermediate filaments lining the inner nuclear membrane; lamin B (68kD), lamin A (74kD), and lamin C (60kD))Lamina associated proteins (LAP 1A and LAP 2) also give continuous rim staining of the nuclear envelope.


These antibodies occur in patients with PBC, AIH, chronic fatigue syndrome and viral hepatitis type D.

References 46, 47, 48, 49, 50

photo 42




Autoantibodies recognizing nuclear membrane related antigens

Anti-nuclear pore complex antibody

Pattern Nuclear membrane (pores) staining, coarse nuclear staining associated with nuclear rim staining in interphase cells.

Antigen gp210 (210kD glycoprotein organizing nuclear pores). The antigenic epitope of this protein is located in its cytoplasmic tail, and also epitopes in its carbohydrate moiety or amino terminal domain directed to the lumen of the pore were reported.


PBC


photo 43




Autoantibodies recognizing antigens of cytoplasmic organelles

Fluorescence patterns of cytoplasmic autoantigens

There are many important organelles in the cytoplasm which fulfill various functions of the cell as described in section 1. Various autoantibodies, known as anti-cytoplasmic antibodies, targeting the proteins organizing these organelles and many cytosol proteins have been found.

■ Autoantibodies recognizing antigens of cytoplasmic organelles

Anti-mitochondrial antibody

Pattern Coarse granular filamentous staining extending throughout the cytoplasm.

Antigen Anti-mitochondrial antibodies-M2 (AMA-M2): pyruvate dehydrogenase (PDH-E2), located on the inner mitochondrial membrane. Antibodies to AMA-M1 and sub-patterns of AMA-M3 to M9 were reported. The staining shown here is due to AMA-M2 antibodies.


IF (rodent kidney or stomach sections used as substrates), ELISA, WB, CLEIA


These antibodies occur frequently in patients with PBC and their presence constitutes one of the diagnostic criteria of the disease. Recently, the presence of mitochondrial autoantigen in bile was reported.

References 53, 54

photo 44 photo 45Staining of AMA-M2 using rodent kidney and stomach sections as substrates. Cytoplasmic staining in the renal tubular cells (intense staining in the distal tubular cells) and intense fluoresence in the parietal cells of the stomach are observed.





Anti-ribosomal antibody

Pattern Homogeneous cytoplasmic staining with perinuclear accentuation is associated with nucleolar staining.

Antigen 3 phosphoproteins of 60S ribosomal particles (P0(38kD), P1(19kD), and P2(17kD)), 28S ribosomal RNA, L12 protein


IF, DID, WB, Immunoprecipitation


These antibodies occur in patients with SLE associated with neuropsychiatric symptoms. Presence in scleroderma patients was also reported.


photo 46 photo 47Staining of anti-ribosomal antibody using rodent kidney and stomach sections as substrates. The diffuse staining in the kidney tubular cells and fluorescence of the chief cells of the stomach are observed. (Comparatively, anti-mitochondrial antibodies fluoresce in the pariental cells of the stomach.)





Anti-Jo-1 antibody

Pattern Fine granular speckled cytoplasmic staining, but weak fluorescence is usual. The granules are condensed around the nucleus and diminish toward the periphery of the cell. The reference serum of AF/CDC-10 from CDC rarely stains in the usual dilution of the test. It was reported that Jo-1 antigens exist in both nucleus and cytoplasm.

Antigen Histidyl-tRNA-synthetase (catalyzing the binding reaction of histidine to 3'-OH of tRNAHis)


DID, ELISA, Immunoprecipitation, CLEIA


These antibodies occur specifically in patients with PM/DM, and is frequently associated with interstitial pulmonary fibrosis and polyarthritis. The presence of these antibodies consititutes one of diagnostic criteria for PM/DM.

References 2, 58

Antibodies to other aminoacyl-tRNA-synthetases (described below) showing similar staining patterns to anti-Jo-1 antibody are found in patients with polymyositis and dermatomyositis. [Reference: 59,74, 75]Anti-PL-7 antibody … threonyl-tRNA synthetaseAnti-PL-12 antibody … alanyl-tRNA synthetaseAnti-OJ antibody … isoleucyl-tRNA synthetaseAnti-EJ antibody … glycyl-tRNA synthetaseAnti-KS antibody…asparaginyl-tRNA synthetase

Antibodies to the signal recognition particle (SRP) show fine dense speckled cytoplasmic staining, and is highly specific to polymyositis. The targeting 54kD protein of SRP bind to the rough endoplasmic reticulum and ribosomes. In patients with these antibodies, poor response to therapy and frequent recurrence are characteristically observed.[Reference: 60]

photo 48AF/CDC-10*





Anti-lysosomal antibody

Pattern Large speckles distributed throughout the cytoplasm fluorescence.

Antigen Lysosomal proteins. The enzymes in lysosomes like cathepsin and lysosome-associated membrane proteins (LAMPs) are considered as candidate antigens. However, they are poorly characterized.


These antibodies occur infrequently in patients with SLE. Clear clinical associations are not known.

References 1, 2

Anti-Golgi apparatus antibody

Pattern Irregular discontinuous granular staining in cytoplasm around the nucleus.

Antigen 230kD protein of Golgi apparatus. Recently 97kD (golgin-97) and 200kD non-myosin proteins were reported.


These antibodies occur infrequently in patients with SLE and SS. The Golgin-97 was reported to be specific to secondary SS.

References 61, 62

photo 49

photo 50





autoantibodies recognizing the protein components of various organelles in cytoplasm

The photos shown below suggest the presence of autoantibodies recognizing the protein components of various organelles in cytoplasm, the enzymes in vesicles and secretory granules or degraded substances taken into the vesicles. They show dense-to-coarse cytoplasmic staining, but the corresponding antigens are not determined. Photo 54 shows the pattern relevant to cell cycle.

photo 51Dense staining throughout cytoplasm.

photo 53Similar staining to the mitochondrial antibodies but the speckles are different.

photo 52Same as photo 51, but more coarse staining.

photo 54Some of the cell populations show cytoplasmic staining.



Autoantibodies recognizing mitotic spindle apparatus related antigens

■ Autoantibodies recognizing mitotic spindle apparatus related antigensThe main component protein of the microtubles of the spindles is tubulin, and the microtubules bind to motor proteins like kinesin and dynein. Anti-spindle apparatus antibodies like NuMA antibodies possibly recognize the component proteins and the binding proteins of the microtubules.

Anti-spindle apparatus antibody

Pattern The spindle fibers between the poles are stained in mitotic cells.

Antigen Tubulin


The presence of anti-tubulin antibodies were reported to occur in patients with parasitic infections and those with Guillain-Barre syndrome.

References 63, 64

Anti-nuclear mitotic apparatus (NuMA)-1 antibody

Pattern The spindle poles and the spindle fibers around them are stained in mitotic cells. Nuclear speckled staining can be seen in interphase cells.

Antigen 210kD protein

Other analytical methods

WB


These antibodies occur in patients with autoimmune diseases like SS.

Reference 65

photo 55

photo 56




Autoantibodies recognizing mitotic spindle apparatus related antigens

Anti-nuclear mitotic apparatus (NuMA)-2 antibody

Pattern The spindle poles and the spindle fibers are stained in mitotic cells. At anaphase/telophase, the staining is hifted to the intercellular bridge and the midbody. The nucleus and cytoplasm show no staining in the interphase cells.

Antigen kinesin-like protein HsEg5(130kD)


Immunoprecipitation


These antibodies occur in patients with autoimmune diseases like SLE.

References 65, 66

Anti-centriole antibody

Pattern The poles of the mitotic spindle are stained in the mitotic cells and one or two dots are stained in the cytoplasm of the interphase cells.

Antigen 48kD heat shock proteins (enolase)


The antibodies occur infrequently in patients with Raynaud's syndrome and SSc.

References 67, 68

photo 57

photo 58



Autoantibodies recognizing cytoskeleton related antigens

■ Autoantibodies recognizing cytoskeleton related antigensCytoskeletons are comprised of three kinds of fibrous proteins; microfilaments (5 to 6 nm in diameter), intermediate filaments (10 nm) and microtubules (25 nm). The microfilaments consist of actin and various binding proteins. The intermediate filaments in HEp-2 cells comprise cytokeratin and vimentin. The microtubules consist of tubulins and organize the cell division apparatus.

Anti-actin antibody

Pattern Microfilaments are stained throughout the cytoplasm.

Antigen Actin


IF, ELISA

C l i n i c a l significance

These antibodies occur in patients with AIH.

References 69, 70

The autoantibodies to actin binding proteins, tropomyosin and vinculin give similar fluorescence to those given by anti-actin antibodies. [References 1]


photo 59 photo 60Staining using rodent kidney and stomach sections as substrates.The vascular walls and the basement membrane of the glomeruli are stained and the muscular layer and muscularis mucosae of the stomach are stained.



Autoantibodies recognizing cytoskeleton related antigens

Anti-cytokeratin antibody

Pattern Reticular cytoplasmic staining (weak to intense fluorescence depending on the cells) and granular staining in mitotic cells are seen.

Antigen Cytokeratin (40 to 52kD intermediate filament)


WB


These antibodies are frequently observed in patients with RA and also occur in other diseases. The presence of antibodies in early RA patients with negative rheumatoid factor was reported.

References 71

Anti-vimentin antibody

Pattern Cobweb-like cytoplasmic staining with denser perinuclear staining.

Antigen Vimentin (53kD intermediate filament)


WB


The antibodies occur in patients with RA, liver diseases and chronic fatigue syndrome.

References 48, 72

photo 61

photo 62 photo 63Staining using rodent kidney and stomach sections as substrates.The vascular wall of kidney and stomach mucous membrane are stained, but it is different from the staining produced by anti-actin antibodies.



Other fluorescence patterns

Other fluorescence patterns

■ Discussion about staining of “rods and rings (RR)” in Fluoro HEPANA test (HEPANA)

In our sample staining service for the customers using HEPANA, there are specific patterns which look like “rod-type” and “ring-type “fluorescent staining in a cytoplasm. These are called “cytoplasmic rods and rings (RR)” as reported by several papers. In recent knowledge, it became clear that RR is detected specifically in serum of patients with chronic hepatitis C. Among them, RR is detected more frequently in hepatitis C patients receiving combination therapy of pegylated Interferon (PEG-IFN) and ribavirin (RBV). It also seems that so far, there are no reports that RR was detected in patients without any treatment. The current study reports CTPS1 and IMPDH2 were identified as components associated with RR.

CTPS1 and IMPDH2 play role in the biosynthetic pathway for CTP and GTP, respectively, which are as nucleotide sources for DNA/RNA. It is known that RR appears when the CTP or GTP synthetic pathway is inhibited, and it is also reported that RR formation was observed even when HEp-2 cells themselves were treated with CTPS1 or IMPDH2 inhibitor. The ribavirin is an IMPDH2 inhibitor, so that RR may be formed in hepatitis C patients who were actually treated with ribavirin. If the formed RR was released somehow into the blood, the immune system would recognize it as a foreign substance to induce autoantibodies to RR. This is the assumed mechanism of RR staining detected by sera from patients with hepatitis C. As possible mechanisms for formation of RR in HEp-2 cells, it is reported that RR was formed by not only inhibition of CTP and GTP synthetic pathway, but also other conditions, such as nutrient starvation (lack of glucose), addition of sodium azide, and treatment with some kinds of kinase inhibitor. In case of our HEPANA, MBL has experience that the frequency of RR formation is dependent on the lot of the calf serum used for HEp-2 cell culture. Such lot-to-lot variation may provide different nutritional conditions to form RR. In fact, HEp-2 growth rate seems to be variable according to the lot of calf serum. Together with, we assume that when HEp-2 cells are exposed with a certain lot of calf serum which tend to provide a starvation, RR formation would be induced in the cells. [reference76-80]



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