In vivo cell penetration and intracellular transport of anti-Sm and anti-La autoantibodies

International Immunology, Vol. 12, No. 4, pp. 415–423

© 2000 The Japanese Society for Immunology

In vivo cell penetration and intracellular transport of anti-Sm and anti-La autoantibodies Sophie X. Deng, Elaine Hanson and In˜aki Sanz Department of Immunology and Microbiology, and Department of Medicine, University of Rochester Medical Center, 601 Elmwood Avenue, Box 695, Rochester, NY 14642, USA Keywords: autoantibody, autoimmune disease, intracellular antibodies, ribonucleoproteins, systemic lupus erythematosus

Abstract Anti-nuclear autoantibodies (ANA) are the hallmark of systemic autoimmune diseases. Yet, the in vivo function of ANA remains controversial to a large extent due to the intracellular nature of their antigenic targets. It has been reported that a subset of autoantibodies can penetrate live cells and translocate into the subcellular compartments containing the corresponding antigens. The studies presented herein show that murine anti-Sm and anti-La monoclonal autoantibodies can also enter a variety of cell types from different animal species and that the cell penetration activity is not isotyperestricted. Interestingly, only mAb with cross-reactivity against double-stranded DNA did enter cells. Both these autoantibodies rapidly accumulate in the nucleus of viable cells but display different penetration kinetics. In co-localization experiments, monoclonal autoantibodies did not accumulate significantly within endocytic vesicles containing dextran, suggesting that they are internalized by mechanisms distinct from conventional receptor-mediated endocytosis. This report represents the first evidence that anti-La and anti-Sm autoantibodies are capable of entering live cells. Our observations support the notion that the phenomenon of intracellular autoantibodies may have a larger scope than previously reported and are consistent with a potential pathogenic role for ANA. Introduction Anti-nuclear antibodies (ANA) have been used to define systemic autoimmune diseases for decades, often with great specificity for the disease in question. As a result, ANA constitute important diagnostic tools for a number of diseases, e.g. systemic lupus erythematosus (anti-Sm and anti-double-stranded DNA antibodies), generalized systemic sclerosis (anti-DNA topoisomerase I antibodies) and localized systemic sclerosis (anti-centromere antibodies) (1–3). A cardinal feature of ANA is that their antigenic targets usually carry out critical intracellular functions such as DNA and RNA processing and modification, protein synthesis, and cell cycle regulation (4–6). It is also of great significance that the recognized epitopes are highly conserved in evolution and localized in domains of functional importance. As a consequence, autoantibodies have been shown to be capable of inhibiting antigen function both in vitro and in vivo (7). At the same time, the very nature of the autoantigens raises a number of significant questions regarding the ability of autoantibodies to interfere with in vivo functions in a manner relevant to disease pathogen-

esis. Indeed, given the subcellular localization of the autoantigens and the systemic nature of the diseases in question, pathogenic autoantibodies should be able to enter live cells in multiple tissues, reach the appropriate intracellular compartment and interfere with cell function. The ability of polyclonal IgG anti-ribonucleoprotien (RNP) autoantibodies isolated from autoimmune patients to penetrate live human peripheral blood mononuclear cells via Fc receptors was first proposed 20 years ago (8). Ever since, an increasing number of autoantibodies of different antigenic specificity have been added to the list of intracellular antibodies (9–19). Yet, it should be noted that only a subset of anti-DNA antibodies has been characterized in some detail for their ability to translocate inside the cell nucleus (16). It should be noted, however, that other investigators have disputed this phenomenon and, indeed, it has been argued that nuclear localization of autoantibody represented a fixation artifact, with movement of Ig into cells during fixation (20). In order to clarify this issue, we have investigated the scope and

Correspondence to: I. Sanz Transmitting editor: C. Martinez-A

Received 20 May 1999, accepted 30 November 1999

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Fig. 1.

Fig. 2

Intracellular autoantibodies 417 nature of intracellular autoantibodies. Here we describe two murine monoclonal autoantibodies with anti-Sm (IgA) and antiLa (IgG) primary antigenic reactivity respectively, which are

capable of penetrating live cells. This represents the first description of monoclonal autoantibodies of such specificity with in vivo nuclear localization ability. Our data indicate that the phenomenon of cellular penetration by autoantibodies has a larger scope than previously suspected and that ANA may indeed contribute to the pathogenesis of autoimmune diseases. The studies also contribute preliminary information regarding the utilization of distinct mechanisms of cell entry and/or intracellular transport of autoantibodies. Methods Antibodies origin and purification methods Anti-La and anti-Smith monoclonal autoantibodies were kindly provided by Dr Stephen Clark (University of North Carolina at Chapel Hill). They were originally isolated from MRL/lpr⫹/⫹ mice and have been described elsewhere (21–23). Antibodies from cell culture supernatants were precipitated with 47% w/v ammonium sulfate and dialyzed in high salt buffer containing 3 M NaCl/10 mM Na borate (pH 8.9). Antibodies of IgA isotypes were affinity-purified using an anti-mouse κ agarose column (Zymed, San Francisco, CA), whereas IgG autoantibodies were purified using Gammabind beads (Pharmacia, Piscataway, NJ) according to the manufacturer’s instructions. All purifications were done under high salt conditions in order to achieve antigen dissociation. IgA and IgG isotype controls were purchased from Sigma (St Louis, MO). Cell lines

Fig. 3. Internalization kinetics of monoclonal autoantibodies in H35 cells. (a) Import kinetics of mAb21 in H35 cells was studied using flow cytometry. In vivo nuclear localization assays were performed as in Fig. 1. Intracellular accumulation of mAb21 was detected with Alexa 488-conjugated secondary antibody and measured as mean fluorescence intensity. mAb 21 was detectable inside the cells within minutes of incubation and reached plateau within 1 h. In contrast, neither intracellular mAb 2-12 nor an IgG2a isotype control were detected. (b) Kinetics of nuclear import of mAb 5B5 was determined by direct measurement of nuclear fluorescence using a CCD camera. Nuclear accumulation of 5B5 was maximal after 30 min of incubation with live cells. Consistent with multiple other experiments, the IgA isotype control was not detected in the nucleus.

Three cell lines previously shown to be susceptible to antibody penetration were selected for analysis. They included H35, a rat hepatoma cell line (16), PK15, a pig kidney cell line (24), and A431 (25), a human epidermoid carcinoma cell line, which were purchased from ATCC (Rockville, MD). Cells were grown and maintained in 10% FCS in DMEM supplemented with 1 mM L-glutamine, Na pyruvate and non-essential amino acids (Gibco/BRL, Gaithersburg, MD). ANA assays Target cells were cultured to near confluence as described above. Cultures with at least 95% cell viability were used for ANA determination. Cells were washed 3 times with PBS (pH 7.4) and fixed with cold acetone/methanol at –20°C for 10 min. After an additional 2⫻PBS washes, the fixed cells were blocked with 5% normal goat serum (Jackson Immuno-Research, West

Fig. 1. ANA and in vivo nuclear localization activities of anti-Sm and anti-La murine monoclonal autoantibodies. ANA activity of Alexa 488conjugated mAb and control polyclonal IgA were tested by conventional indirect immunofluorescence (upper panel). mAb were incubated with acetone/methanol-fixed H35 cells. Bound antibody was then labeled with Alexa 488-conjugated goat anti-mouse IgG (H ⫹ L). All three mAb, 21, 5B5 and 2-12, generated distinct ANA patterns but the IgA isotype control was seen only in the cell membrane and in the cytosol. The right panel shows results obtained with the in vivo nuclear localization assay (lower panel). In this assay only mAb 21 and 5B5 were detected inside the nucleus. Images were obtained by conventional fluorescence microscopy (⫻40). Fig. 2. Nuclear localization of mAb as shown by confocal microscopy. In vivo nuclear localization experiments were performed as in Fig. 1 with mAb 21, 5B5 and 2-12 as well as with a polyclonal IgA control antibody. The cell nucleus was labeled with propidium iodide 0.5 µg/ml. Samples were scanned individually under FITC (left panel) and propidium iodide settings (middle panel) using a BioRad MRC 600 confocal microscope (⫻100). The merged images (right panel) show that only mAb 21 and 5B5, but not 2-12 or IgA isotype control co-localize with propidium iodide-stained material within the cell nucleus. The IgA isotype antibody control can be observed in the cytosol, presumably due to pIgR-mediated endocytosis, but not within the nucleus.

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Fig. 4. Cell lineage susceptibility to autoantibody penetration. Penetration of mAb 21 and 5B5 was demonstrated using cell lines of different tissue origins derived from several animal species. Both monoclonal autoantibodies were able to penetrate a human epidermoid carcinoma cell line (A431) and a pig kidney cell line (PK15) as determined by in vivo nuclear localization assays performed as previously described for H35 cells. Appropriate antibody controls (mAb 2-12, IgA and IgG2a isotype controls) did not enter the cells (not shown). Images were taken using a conventional fluorescence microscope (⫻40).

Fig. 5A.

Intracellular autoantibodies 419 Grove, PA) for 10 min. ANA were then detected with Alexa 488conjugated goat anti-mouse IgG (H ⫹ L) (Molecular Probes, Eugene, OR) for 30 min at room temperature and analyzed by conventional indirect immunofluorescence microscope. In vivo cell penetration assay Penetration assays were modified from previously published protocols (14,16). mAb H7 and H9, previously shown to possess in vivo intracellular activity, were kindly provided by Dr Michael Madaio (University of Pennsylvania, Philadelphia, PA) (16) and used as positive controls in our initial experiments. Briefly, cells were cultured at a concentration of 5⫻105/ml in Lab Tek chamber slides (Nalge Nunc, Naperville, IL) and used when they had reached at least 80% confluence. At that point, cells were washed twice with 1% BSA/PBS and incubated with the appropriate mAb (30 µg/ml) in 1% BSA/DMEM for a predetermined period of time. Cells were then extensively washed and their viability was assessed by their ability to exclude vital dyes such as Trypan blue or propidium iodide. In order to ensure that any intracellular antibody observed was indeed the result of in vivo penetration that occurred prior to fixation, the cells were snap-fixed in pre-chilled acetone/methanol at –20°C for 10 min. Alternatively, cells were fixed using different, freshly prepared cross-linking agents including 4% paraformaldehyde for 1 h at room temperature and 3% formaldehyde on ice for 15 min followed by permeabilization with 0.2% Triton X-100 for 5 min on ice. After 3 washes with PBS, intracellular antibodies were detected using Alexa 488-conjugated secondary antibodies as described above. IgA antibodies were also labeled using the same secondary antibody which also recognizes κ light chains. For co-localization experiments, the nucleus was co-stained with propidium iodide (0.5 µg/ml) for 10 min at room temperature. Results were then visualized under an Olympus BX60 fluorescent microscope (Tokyo, Japan) or scanned using a BioRad MRC 600 confocal microscope (Hercules, CA). Internalization kinetics For mAb 21 kinetics, H35 cells were detached from the tissue culture dish by incubation with 10 mM EDTA/PBS (pH 8.0) and cell penetration was assayed at different time points as described above. These experiments were performed either at 37°C or, alternatively, at 4°C by keeping the target cells and all reagents on ice previous to the incubation experiments which were then carried out in the cold-room at temperatures ranging

between 0 and 4°C. Cell viability was monitored by Trypan blue exclusion and was consistently ⬎90% at the time of fixation. The amount of internalized antibodies was measured as mean fluorescence intensity using an Elite flow cytometer (Coulter, Fullerton, CA). Nuclear localization kinetics of mAb 5B5 was determined by indirect immunofluorescence as described above for in vivo penetration assays. Nuclear fluorescence was then quantified from digital images obtained with a Sony DXC9000 3CCD camera (Tokyo, Japan) using the Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Six fields were counted from each sample in at least three independent experiments Fluid-phase endocytosis experiments Intracellular transport of mAb was compared to trafficking of Texas Red–dextran. To do so, H35 cells were incubated with antibodies at 30 µg/ml and Texas Red–dextran (70,000 kDa) at 1 mg/ml (Molecular Probes) in 1% BSA/DMEM for 30 min at 37°C. After 3 washes with PBS, cells were fixed with cold acetone/methanol and intracellular mAb were detected with Alexa 488-conjugated goat anti-mouse IgG (H ⫹ L) as described for the penetration assay. Samples were scanned using a Leica confocal microscope (Heerbrugg, Switzerland) to obtain images of Alexa 488 and Texas red labeling respectively. Image processing was performed with Adobe Photoshop software (San Jose, CA). Both granules containing only mAb, and those containing dextran and mAb together were counted. Results were expressed as the fraction of total mAb granules that also contained dextran. A final score was generated from counting a minimum of three fields from three independent experiments. Antibody sequence analysis The DNA sequences of the mAb have been previously reported and were obtained from GenBank (accession nos X67195, L08989, L08980, U26999, L09013, U18595, S71117 and S71116). The sequences were imported into the EditSeq program of the DNASTAR Lasergene software (Madison, WI) and analyzed for VH and VL germline homology, and the presence and pattern of somatic mutations using the Megalign program of DNASTAR and the Blastn search program available through the National Center for Biotechnology Information. Analysis of the predicted protein sequences was performed using the Protean program of DNASTAR.

Fig. 5. Co-localization of intracellular autoantibodies and endocytic vesicles. (a) Texas Red–dextran (1 mg/ml) and the corresponding mAb were co-incubated with live H35 cells at 37°C for 30 min. Intracellular antibody was labeled with Alexa 488-conjugated secondary antibody as before. Samples were scanned using a Leica confocal microscope (⫻100) under the appropriate settings in order to detect Texas Red (right panel) or Alexa 488 (middle panel). Individual images of the same field were merged to detect colocalization of mAb and dextran (right panel). As compared to endocytosed IgA control, only a small percentage of vesicles containing mAb 21 or 5B5 also contained dextran. Quantification of endocytosis vesicles containing both mAb and dextran. (b) Results from the experiments shown in (5) are expressed as the percentage of Alexa 488⫹ vesicles also containing Texas Red.

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Fig. 6. Sequence analysis of the HCDR3 of mAb. The predicted amino acid sequences of both penetrating (mAb21 and 5B5) and nonpenetrating mAb (1-12 and 2-12) are aligned. Arrows indicate the areas corresponding to the end of the FR3 as well as conventional HCDR3 regions. Also, the extended HCDR3 loop containing the antigen contact residues is indicated in the figure. The last residue shown in the figure (W) corresponds to the beginning of the FR4 for all sequences. Basic residues are shown in bold italics and the RR doublet unique to penetrating antibodies is encased.

Results In vivo penetration of H35 cells by mouse monoclonal autoantibodies In order to explore the prevalence of the intracellular autoantibody phenomenon and given that the preponderance of previous studies had focused on anti-DNA antibodies, we decided to test a panel of disease-associated monoclonal autoantibodies whose primary specificity was other than DNA. Moreover, we wanted to determine whether this phenomenon is restricted to any particular isotype. Thus, four anti-Sm and one anti-La mAb derived from autoimmune MRL/lpr⫹/⫹ mice were selected for our in vivo penetration assay. These mAb were previously characterized and have been described elsewhere (21–23). In selecting these autoantibodies we were mindful of the fact that anti-RNP autoantibodies often display significant crossreactivity with single-stranded DNA and/or double-stranded DNA. Therefore, we chose to study antibodies with different degrees of cross-reactivity with DNA. Furthermore, it has been suggested that pathogenic anti-DNA antibodies may predominantly cross-react with Sm/RNP-associated polypeptides A and D (24). Accordingly, we selected anti-Sm antibodies with different degrees of reactivity against either the D or the B/ B⬘ polypeptides of the Sm antigen, which represent the main antigenic targets of the anti-Sm response. Relevant characteristics of these mAb including in vitro ANA pattern and in vivo penetration ability are summarized in Table 1. As expected, all mAb showed in vitro ANA activity. As shown in Fig. 1, among those five mAb, only two mAb, 21 (anti-La, IgG2a) and 5B5 (anti-Sm, IgA), were detected in the nucleus of live H35 cells. In order to ensure that nuclear localization of mAb was not due to leakage of cell and nuclear membranes, cell viability was monitored by Trypan blue or propidium iodide exclusion before fixation in all experiments (data not shown). The in vivo nuclear accumulation of mAb was not affected by different fixatives including cross-linking reagents such as freshly prepared 4% paraformaldehyde or methanol-free 3% formaldehyde (data not shown). Significant similarities could be observed between the ANA patterns and the in vivo staining profiles. Thus, mAb21 created a nuclear rim pattern in both assays while a consistent speckled pattern was seen with 5B5. In addition to the nuclear localization, granules containing mAb 21 were also observed in the cytoplasm of most H35 cells and somewhat finer ones with mAb 5B5. Large cytoplasmic granules were also observed with the IgA isotype control antibody, but in the absence of nuclear staining. This observation

might be explained by endocytosis of IgA via the polymeric IgA receptor (pIgR) that mediates transcytosis in hepatocytes (26–29). Taken together, these results strongly suggest that the cell entry and nuclear localization of the mAb is Fc receptor independent (as demonstrated by lack of activity of other mAb of the same isotype and of polyclonal IgG and IgA antibodies). Moreover, the consistently positive results obtained with mAb 21 and 5B5 under different fixation protocols along with the negative controls just discussed provide further proof that this phenomenon is not the result of fixation artifacts. The staining profile obtained with mAb21 suggested that this antibody might simply bind to the nuclear membrane without actually entering the nucleus. This issue was resolved by using laser scanning confocal microscopy to show that both mAb 21 and 5B5 did indeed co-localize with propidium iodide-stained material within the cell nucleus (Fig. 2). It should be appreciated, however, that within the nucleus, there was only partial overlap between the two probes, consistent with the recognition by the mAb of nuclear antigens other than or in addition to double-stranded DNA. This is not surprising in light of their primary antigenic reactivity against the Sm and La proteins respectively. In vivo penetration kinetics Both mAb, 21 and 5B5, penetrated live cells in a time-dependent manner. Internalization kinetics of mAb 21 was determined by flow cytometry. Cell aliquots sampled at each time point were also observed under fluorescent microscopy to confirm nuclear localization of the antibody. In turn, mAb 5B5 presented a different challenge based on its IgA isotype. As previously shown, IgA antibodies are internalized by H35 cells presumably through the pIgR and, therefore, mere cell penetration as indicated by flow cytometry would not be indicative of nuclear localization. Accordingly, 5B5 kinetics was determined by direct measurement of nuclear fluorescence intensity from the digital images obtained by a CCD camera. As shown in Fig. 3, mAb 21 and 5B5 entered live cells in a very rapid manner, and could be detected in the cell nucleus within 15 min of incubation. Penetration of mAb 21 reached a plateau between 30 and 60 min, while maximal nuclear staining with mAb 5B5 was seen within 30 min. Furthermore, the maximum nuclear accumulation of mAb 21 was significantly higher than that of mAb 5B5 when both antibodies were assayed under fluorescent microscopy (data not shown). These results may reflect differences between both mAb in terms of efficiency in cell

Intracellular autoantibodies 421 penetration or differences in the efficiency of nuclear import and/or export. Also of note, no significant differences in cell penetration or nuclear accumulation were observed when the same experiments were performed at temperatures of 0–4°C, which are expected to significantly interfere with active transport mechanisms (results not shown). Cell type susceptibility to autoantibody penetration Along with H35, two additional cell lines, A431, a human epidermoid carcinoma, and PK15, a pig kidney cell line, were tested to determine their susceptibility to autoantibody penetration. As shown in Fig. 4, mAb 21 and 5B5 were also able to enter both cell types. As with H35, nuclear localization was found only with these autoantibodies but not with IgA or IgG isotype controls or with a negative autoantibody control mAb 2-12. Nuclear localizing mAb utilize distinct intracellular transport mechanisms As a preliminary attempt to characterize the mechanism(s) of autoantibody uptake and transport, we asked whether the mAb could be found in endocytic vesicles. We used dextran, an indicator of fluid phase endocytosis (30,31), to track the endocytic pathway. As shown in Fig. 5, only a relatively small fraction of vesicles contained both mAb and dextran together (27.4 and 18.7% for mAb 5B5 and mAb21 respectively). In contrast, ⬎85% of dextran-positive vesicles also carried the IgA isotype control antibody. The scattering pattern of the cytoplasmic IgA– dextran vesicles is consistent with them being contained by endosomes. The remaining 15% of vesicles likely represents transcytotic vesicles carrying post-endosomal IgA molecules. Discussion The actual role and mechanism(s) of action of anti-nuclear autoantibodies in the systemic autoimmune diseases are still questionable 50 years after the discovery of the ANA phenomenon (32). The most widely accepted pathogenic mechanism is the formation of circulating immune complexes with inflammatory properties and indeed such mechanism has been well documented with anti-DNA antibodies. Given the intranuclear nature of most of the autoantigens recognized, it seems obvious that the ability of autoantibodies to enter subcellular compartments would create additional and tantalizing opportunities for these molecules to generate functional perturbation and tissue damage. This report sheds light on this poorly understood aspect of the autoimmune process. Our results demonstrate that, in addition to DNA, other major nuclear autoantigens such as the Sm and La RNP can also be the target of intracellular autoantibodies. First, it should be emphasized that this property was observed in only a subset of the autoantibodies tested and that it was independent of the type of fixatives used, either acetone or cross-linking agents including 4% paraformaldehyde. This result indicates that our observations are not the result of fixation artifacts with disruption of the plasma membrane and/or the nuclear envelope as it has been suggested elsewhere (20). Also of importance, both the absence of Fc receptors in the cell lines tested as well as the lack of internalization of isotype control antibodies strongly suggest that this phenomenon is Fc receptor independent and is most likely

mediated by the antigen binding site of the antibodies tested. Being a liver epithelial cell line, however, H35 cells do express the pIgR that normally mediates endocytosis and transcytosis of both dimeric IgA and pentameric IgM. Yet, as demonstrated by using a control polyclonal IgA antibody, internalization of IgA through the pIgR does not result in nuclear translocation, thus reaffirming the specificity of this phenomenon for the IgA mAb with anti-Sm reactivity (5B5). Several factors related to the antigenic reactivity of mAb 21 and 5B5 might be responsible for their intracellular activity. First, anti-Sm and anti-La antibodies may display significant cross-reactivity with single-stranded and/or double-stranded DNA, and, therefore, it is plausible that such cross-reactivity might be ultimately responsible for cell penetration. In our sample, only autoantibodies with cross-reactivity with doublestranded DNA but not single-stranded DNA were able to enter live cells (see Table 1) suggesting that double-stranded DNA binding may be important for antibody penetration. Secondly, anti-Sm antibodies may react with one or more of the multiple polypeptides that form the snRNP complex. The mAb tested in this study were chosen to represent a spectrum of anti-Sm reactivity with special emphasis on the main antigenic targets, i.e. the B/B⬘ and/or D polypeptides (21). While mAb 5B5 reacts only with the Sm-D polypeptide, other anti-Sm-D mAb failed to penetrate live cells, as did mAb 1-12 which binds to both SmB/B⬘ and -D polypeptides. Hence, on a superficial analysis it would seem that the global polypeptide reactivity of these autoantibodies is not essential, or at least not sufficient, for cell entry. It is possible, however, that the intracellular properties could be dependent on fine epitope specificity in which case the broader reactivity against the full-length polypeptides might not be informative enough (detailed epitope mapping of the anti-Sm mAb is currently underway). Along similar lines, the fine specificity of anti-La antibodies might also represent an important factor in their ability to translocate into the cell nucleus. The demonstration that the ability of anti-La antibodies to recognize different La–RNA complexes is dependent on their fine specificity would appear to support this notion (33,34). Alternatively, intracellular autoantibodies might need to crossreact with surface-exposed antigens as it has been suggested for some anti-DNA antibodies that recognize myosin I (35). Although our antibodies did not recognize myosin I as determined by conventional ELISA (results not shown), other possible targets include laminin, a main component of basal membranes that is recognized by some pathogenic anti-La antibodies capable of inducing congenital heart block (36). Another potential scenario contemplates the possibility that the intracellular antigens themselves could be expressed in the cell membrane, and, indeed, both La and the snRNP have been shown to translocate to the cell surface under diverse stimuli including UV radiation, estrogen stimulation, viral infections and exposure to tumor necrosis factor-α (37–41). Whether the antigen responsible for autoantibody penetration turns out to be the nominal antigen (Sm or La) or a crossreactive antigen(s) such as DNA, our results suggest that the recognized target must be a widely expressed molecule since several cell lines including rat liver cells, pig kidney cells and human epidermoid carcinoma cells were susceptible to antibody penetration. This finding is consistent with the observations of Vlahakos et al., that the in vivo pathogenic potential of

422 Intracellular autoantibodies a subset of anti-double-stranded DNA antibodies administered to normal mice correlated with their ability to accumulate in the cell nucleus in multiple tissues (16), and it supports the notion that anti-Sm and anti-La autoantibodies could mediate systemic autoimmune tissue damage. The mechanism responsible for intracellular transport of mAb remains largely unexplored and may not correspond to the general pathways of protein kinesis. Our results represent a significant departure from the observations reported with the subset of anti-DNA autoantibodies that cross-react with myosin which are internalized by receptor-mediated endocytosis (35). Thus, in our case nuclear accumulation was faster with significant nuclear staining observed within minutes. This feature is reminiscent of the rapid cell entry and nuclear translocation observed with the HIV Tat protein. This translocation property has been assigned to a region of the Tat protein centered on a cluster of basic amino acids and recent data have demonstrated that chemical coupling of this Tat-derived peptide to several proteins allowed their internalization into several cell lines or tissues (42). As with our mAb, the rapid uptake of Tat peptides appears to be temperature independent, suggesting that the internalization process does not follow conventional endocytosis rules. Multiple studies have shown that the presence of basic residues in the antibody hypervariable regions, mostly arginine residues in the HCDR3, may be responsible for anti-DNA reactivity (43) and that cationization of otherwise irrelevant antibodies also appears to induce cellular uptake (44). Furthermore, these stretches of basic residues may closely resemble conventional nuclear localization signals (NLS) such as the one used by the SV-40 large T antigen (45,46). Close inspection of the published hypervariable region sequences of mAb 21 and 5B5 did not reveal a typical NLS. However, some distinct features of potential significance can be found in the HCDR3 of these antibodies (Fig. 6). Thus, both HCDR3s display an RR doublet at the 5⬘ end where the second arginine appears to have been generated by junctional diversity and presumably selected by antigen. In the case of mAb21 another amino acid with positive charge potential (histidine) is located one residue away from the RR doublet and contributes to create a significantly positive charge in the HCDR3 of this antibody. In contrast, these features were absent in the non-penetrating antibodies whose HCDR3 global charge was significantly less positive, as the result in some cases of a higher content of acidic residues which may promote binding to the basic snRNP polypeptides (47). In summary, in this paper we demonstrate that autoantibodies whose primary antigenic specificity is directed against the major RNP targets of systemic autoimmune diseases such as systemic lupus erythematosus and Sjo¨ gren’s syndrome can rapidly traverse the plasma membrane of live cells of different tissue origin and translocate into the nucleus. Although crossreactivity with double-stranded DNA may play a role, the actual requirements for this phenomenon remain to be determined, and, indeed, previous results by other investigators and our own studies show that anti-DNA reactivity per se is not sufficient for intracellular activity. The rapid rate of internalization, the lack of significant co-localization with endocytic vesicles and the apparent temperature independence observed in our studies strongly point towards a mechanism different from conventional

receptor-mediated endocytosis. Experiments aimed at the elucidation of these issues are currently underway in our laboratory. The observations presented herein hold significant promise for the study of important biological functions such as intracellular and nucleocytoplasmic transport of proteins as well as for our understanding of systemic autoimmunity. Furthermore, once the requirements for in vivo intracellular activity of (auto)antibodies are established, this information should prove invaluable for the design of antibodies as intracellular therapeutic agents and drug-delivery systems. Acknowledgements We are indebted to Drs M. P. Madiao (University of Pennsylvania) and S. Clarke (University of North Carolina at Chapel Hill) for providing the anti-DNA and anti-Sm/La mAb respectively. We are equally indebted to Dr D. Goldfarb (University of Rochester) for advice and discussions on nuclear transport. We thank Kathy Troughton (University of Tennessee at Memphis) and Hiram Lyon (University of Rochester) for their excellent assistance with confocal microscopy. We are also grateful to Drs F. Young, E. Schwarz and M. Nahm for reviewing the manuscript. This study was supported partially by NIH grants PHS AG14878 and AG14585. S. X. D. is supported by NIH 2T32AI07285 Training Grant and the MD/PhD program at the University of Rochester.

Abbreviations ANA NLS pIgR RNP

anti-nuclear antibody assay nuclear localization signal polymeric IgA receptor ribonuleoprotein

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