Mutagenesis, Vol. 14, No. 2, 227-231,
March 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Cytogenetic study and FISH analysis in lymphocytes of systemic lupus erythematosus (SLE) and systemic sclerosis (SS) patients
Dipartimento di Scienze dell'Uomo e dell'Ambiente, Università di Pisa, Pisa, Italy
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Abstract |
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Systemic lupus erythematosus (SLE) and systemic sclerosis (SS) are autoimmune diseases characterized by the presence of antibodies against ubiquitous self antigens. The presence of clastogenic factors (CF) capable of inducing chromosome breakage has also been reported in the plasma of some patients. We aimed to assess basal frequency of cytogenetic damage in lymphocytes and presence of CF in the plasma of two groups of SLE and SS patients displaying a different antibody status (ACA–/Scl70+ or ACA+/Scl70–), using the micronucleus (MN) assay and FISH analysis with a pancentromeric DNA probe. As compared with controls, we found significantly higher MN frequencies in SS patients, but not in SLE patients. In addition, our data showed a significant prevalence of C–MN in SLE and ACA–/Scl70+ patients and of C+MN in ACA+/Scl70– patients. We observed a positive response in three out of the five CF experiments performed on plasma of SS patients. The three patients whose plasma caused MN induction were subtyped as ACA–/Scl70+, whereas the other subjects had ACAs. The same tests on six SLE patients gave negative results.
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Introduction |
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Systemic lupus erythematosus (SLE) and systemic sclerosis (SS) are autoimmune diseases characterized by the presence of antibodies against ubiquitous self antigens. These diseases occur as the consequence of altered immunoreactions depending on complex interactions between genetic and environmental factors (Theofilopoulos, 1995
). SLE autoantibodies react against nuclear (ssDNA, dsDNA, RNA and snRNP) as well as other different cell constituents (Kotzin and O'Dell, 1995). In the majority of SLE patients the disease shows periods of acute clinical manifestations alternating with inactive or remission stages (Kotzin, 1996). In SS, the presence of antibodies to the centromere or to topoisomerase I (Scl70 antigen) seems to be associated with either a mild clinical subset identified as the CREST variant or a more severe form characterized by progressive skin and visceral organ involvement (Moroi et al., 1980; Tan et al., 1980; McCarty et al., 1983; Maul et al., 1986; Shero et al., 1986). Previous studies concerning the presence of either spontaneous cytogenetic abnormalities or plasma clastogenic factors (CF) in somatic cells of SS and SLE patients produced conflicting results (Khondkarian et al., 1967; Emerit, 1976; Emerit et al., 1980; Emerit and Michelson, 1981; Huet-Warembourg et al., 1982; Palmer et al., 1987; Caporossi et al., 1990; Jabset al., 1993; Severin, 1997). CFs consist of a mixture of different substances, the biochemical nature of which varies depending on their origin. They have been shown to act via oxygen free radical formation (Emerit, 1994). In addition, recent evidence indicates that IgG antinuclear antibodies can enter cells and reach their nuclear target, resulting in cell and organ damage (Levine et al., 1991; Ma et al., 1991; Vlahakos et al., 1992; Koren et al., 1995). The presence of these autoantibodies in the nuclear compartment could also be responsible for DNA damage. The aim of the present paper was to assess both the spontaneous frequency of cytogenetic damage in lymphocytes and the presence of CF in plasma from two groups of SLE and SS patients, using the micronucleus (MN) assay. In order to verify the mechanism underlying MN formation, a FISH analysis with a pancentromeric probe was also carried out in subgroups of the study population.
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Study population
The population studied consisted of 26 individuals affected by SLE (15 patients aged 25–63 years) and SS (11 patients aged 33–70 years) and 18 controls aged 24–70 years. All subjects were females with the exception of two (one SLE patient and one control). None of the subjects had recently received drugs or X-rays, although exposure through diet to other substances that can affect MN frequencies could not be ruled out. SS patients were subdivided into two classes according to the presence of antibodies to the centromere or to topoisomerase I (five ACA+/Scl70– and six ACA–/Scl70+, respectively). All but one of the SLE patients were in the inactive disease phase at the time of the study. Experiments for CF analysis were performed using whole blood of a young, healthy, non-smoking female.
Blood collection and MN analysis
Heparinized venous blood samples were drawn from each individual and phytohaemagglutinin-stimulated, cytochalasin B-blocked lymphocytes were cultured for 72 h in triplicate for assessment of spontaneous MN level and MN analysis by FISH or in duplicate for CF experiments. Culture conditions and harvest protocol were the same as described elsewhere (Migliore et al., 1995) except that cytochalasin B was added at 6 µg/ml (final concentration).
FISH analysis
We used a digoxigenin-labelled 
-satellite DNA probe (Oncor) specific for the centromere of all human chromosomes. Slide pretreatment, hybridization and immunodetection were performed according to the protocol described elsewhere (Scarpato et al., 1996).
CF experiments
A subgroup of 11 patients (six SLE and five SS) and 10 sex- and age-matched controls was selected for CF experiments. Plasma from each individual obtained by centrifugation at 3000 r.p.m. for 15 min was immediately frozen at –20°C until use. In accordance with the Emerit (1990) protocol, CF isolation consisted of plasma ultrafiltration at 3000 r.p.m. for 2 h through a filter with a cut-off of 10 kDa (Centriplus concentrators; Amicon) in order to remove all high molecular mass components. The ultrafiltrate was immediately frozen at –80°C if the experiments could not be performed the same day. Test cultures from the young healthy subject (reference donor) received 0.45 ml ultrafiltrate from the patients and controls. In each CF experiment, the spontaneous frequency of MN from untreated blood cultures of the reference donor was recorded as the negative control. Whenever we observed statistically significant MN induction in patient ultrafiltrate-treated cultures, FISH analysis was performed in the same subjects and in their paired controls.
Slide scoring and statistical analysis
In accordance with standard criteria (Fenech, 1993), MN analysis was performed by scoring 2000 binucleate lymphocytes on coded slides from each donor. MN frequency was expressed as number of micronucleated binucleate cells per thousand cells. For both patients and controls, the clastogenic effect of ultrafiltrates was expressed as a clastogenicity index (CI) according to the following formula: CI = (MNtr – MNctrl)/MNctrl, where MNtr is the MN level of the reference donor after ultrafiltrate treatment and MNctrl is the reference donor basal MN level. A total of 100 MN for each subject were fluorescently analysed on a Nikon Optiphot-2 fluorescence microscope equipped with a triple bandpass filter (Omega Optical Inc.) for simultaneous visualization of TRICT and DAPI fluorescence. MN identification and signal evaluation were performed using an oil immersion 100x objective (final magnification 1000x). Only binucleate cells containing the TRITC signal were considered for MN fluorescence analysis. Thus MN without fluorescent signal or showing a red signal indicated a MN arising from an acentric fragment (C–MN) or the presence of a whole chromosome (C+MN), respectively. Values were expressed as percentage of C–MN or C+MN out of total MN analysed. The effect of diseases on MN frequencies was assessed by multifactor analysis of variance (ANOVA) taking into account age, sex and smoking habit as confounding factors.
In the case of FISH analysis the parameter considered was also normalized for MN frequency. For CF experiments, the CI of each patient was compared with that of the matched control using the t-test for paired data. All statistical elaborations were performed by using the STATGRAPHICS (Statistical Graphics Corp.) software package. For MN fluorescence analysis a CI value was also calculated and statistically elaborated as described above. In this case, C–MN or C+MN percentages substituted for MN frequencies.
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Table I
summarizes the demographic and clinical characteristics of the study population, composed of 15 SLE and 11 SS patients and 18 age- and sex-matched controls. There were only four smoking patients and three smoking controls. SS patients were classified into two groups on the basis of their antibody specificity: ACA+/Scl70– or ACA–/Scl70+. Tables II and III give the ANOVA test results on MN and FISH analysis data for SLE and SS patients, respectively. After normalizing for sex, smoking habit and the age effect, mean MN frequency for SLE patients did not significantly differ from the value for the controls (10.6 ± 3.9 versus 9.1 ± 4.6
), whereas SS subjects showed overall statistically significant (P = 0.007) higher MN levels as compared with the control group (27.4 ± 19.9 versus 9.1 ± 4.6). When the data were viewed separately for each SS subset, we found a similar trend in both groups in comparison with controls, although ACA+/Scl70– individuals displayed the most elevated MN frequency (38.0 ± 24.9). No significant difference was observed between the two patient subgroups. In SLE patients, we observed a statistically significant (P < 0.001) higher average percentage of C–MN as compared with the control group (63.8 ± 5.0 versus 38.7 ± 6.8). The overall proportion of C+MN for SS subjects did not differ from that of controls. We also found a significant difference between ACA+/Scl70– patients and both ACA–/Scl70+ (P = 0.034, 77.2 ± 4.5 versus 50.2 ± 3.7%) and control individuals (P < 0.001, 77.2 ± 4.5 versus 61.3 ± 6.8%). In contrast, ACA–/Scl70+ patients showed a significanty higher percentage of C–MN as compared with controls (P = 0.006, 49.8 ± 3.7 versus 38.7 ± 6.8%; data inferred from the corresponding C+MN values of Table III).
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Results of the experiments on CF are summarized in Table IV
. We observed no MN induction in reference donor lymphocytes that had received ultrafiltrated plasma from the six SLE patients or their paired controls. Only ultrafiltrated plasma from three out of the five SS subjects significantly increased the reference donor basal MN level as compared with the corresponding control experiments. FISH analysis performed in SS subject plasma-treated positive cultures revealed an elevated frequency of C–MN, whereas their matched controls showed an equal proportion of C–MN and C+MN. Interestingly, the three SS MN-inducing patients were characterized as ACA–/Scl70+, whereas the others were ACA+/Scl70–.
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Discussion |
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This study reports the results of cytogenetic analysis in lymphocytes of a group of patients affected by autoimmune diseases (15 SLE and 11 SS). We assessed basal MN level and the mechanism of MN formation using the FISH technique with a pancentromeric DNA probe. We also performed additional experiments in order to evaluate the presence of clastogenic activity in patient plasma.
In our study, MN frequency did not differ between SLE patients and controls. On the other hand, SS subjects showed a statistically significant higher overall MN level as compared with that of controls, especially in the case of ACA+/Scl70– individuals. Even if some of the variability between patients and controls or among each subject group could be explained, at least partially, by dietary factors not taken into account during donor recruitment, our data are in agreement with previous reports showing absence of relevant cytogenetic damage in lymphocytes of SLE individuals (Emerit et al., 1974; Emerit, 1976; Caporossi et al., 1990; Severin, 1997). However, there are conflicting results in the literature regarding the presence of chromosome abnormalities in SS patients (Emerit et al., 1974; Pan et al., 1975; Powell et al., 1986; Romani et al., 1986). This may have been due to clinical differences among the subgroups studied (Jabs et al., 1993). It is noteworthy that in our study, although no MN increase was observed in SLE patients, FISH analysis revealed an excess of C–MN, suggesting the prevalence of chromosome breakage. Furthermore, the degradation of nuclear DNA in lymphocytes of SLE was seen to occur at an increasing rate as compared with healthy controls (Compton et al., 1984). This finding could be explained as the consequence of immunocomplex formation, activating the inflammatory response cells, which in turn release oxygen free radicals. Moreover, it is possible that anti-dsDNA antibodies, one of the major SLE serological markers, could damage DNA if they succeed in reaching their target. Evidence for this hypothesis was provided by Ma et al. (1991). Although these events do not necessarily imply an increase in spontaneous MN frequency, they could partially alter the normal physiological processes regulating MN formation mechanisms.
As regards the SS group as a whole, MN fluorescence analysis reflected the same trend observed in the controls, whereas ACA+/Scl70– patients showed a significantly increased proportion of C+MN as compared with either ACA–/Scl70+ or control subjects. The presence of anticentromere antibodies seems to be related to increasing aneuploidogenic events, as previously reported by Jabs et al. (1993). These findings point to a possible relationship between specific antibody characterization (e.g. specific clinical subgroups) and a particular kind of cytogenetic damage. It is well known that defects in centromere structure and/or function can lead to chromosome malsegregation during anaphase, with consequent chromosome inclusion into a MN (Stone and Sandberg, 1995). Autoantibodies against chromosome constituents could cause alteration by interaction with the corresponding antigen. It has recently been suggested that IgG antinuclear antibodies can enter viable cells using mechanisms similar to those of hormones and growth factors (Foster et al., 1994; Yanase et al., 1994). Immunoglobulins purified from serum containing ACAs microinjected into HeLa cells disrupted centromere functionality, thus inducing aneuploidy (Bernat et al., 1990). The elevated proportion of aneuploidogenic event-derived MN we found in ACA+/Scl70– patients might therefore be due to the presence of such antibodies. Accordingly, antibodies against topoisomerase I could account for the significant excess of C–MN observed in ACA–/Scl70+ subjects. In this context, it is worth pointing out that topoisomerase inhibitors are known to be potent clastogenic agents in in vitro or in vivo systems (Ferguson and Baguley, 1996).
As regards CF experiments, treatment with SLE patient ultrafiltrate plasma did not cause MN induction in reference donor lymphocytes. Our data are in agreement with those of Caporossi et al. (1990), although other studies showed the presence of CF as evaluated by the chromosome aberration test (Emerit et al., 1980; Emerit and Michelson, 1981). Differences in patient group composition could explain the conflicting results. Thus our SLE subjects, similarly to Caporossi's patients, were in the inactive or remission stage of the disease, while the group studied by Emerit was presumably in the active phase. We observed a positive response in three of the five experiments performed on plasma ultrafiltrate of SS patients. The three patients whose plasma caused MN induction were subtyped as ACA–/Scl70+, whereas the other subjects had ACAs. Interestingly, the presence of anti-topoisomerase antibodies is a clinical marker associated with more severe pathological conditions of the SS disease. The progressive involvement of derma and visceral organs characterized by abnormal sclerotic processes could be responsible for the formation of plasmatic clastogenic factors. MN fluorescence analysis confirmed the presence of clastogenic activity, since a significantly elevated percentage of C–MN was observed in two out of the three patients. The lack of a significant increase in MN containing acentric fragments in this patient could be due, at least partially, to a lower CF concentration, since interindividual variations in plasma level of clastogenic compounds can account for differences in chromosome breakage induction. It should be remembered that treatment of reference donor cultures with ultrafiltrate plasma from controls produced neither a significant MN frequency increase nor a change in the expected ratio between spontaneous C+MN and C–MN. In addition, the biochemical nature of CFs could differ according to origin, conceivably as a consequence of the characteristics of the two diseases.
In conclusion, our study would indicate the presence both of cytogenetic alterations in lymphocytes and CF in plasma from patients affected by two autoimmune diseases. Our findings contribute to a better understanding of the possible relationship betweeen cytogenetic anomaly and pathological conditions.
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Acknowledgments |
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We are grateful to Drs Paola Migliorini and Daniele Chimenti (Dipartimento di Medicina Interna, University of Pisa) for blood sampling of patients.
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Notes |
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1 To whom correspondence should be addressed. Tel: +39 50 551217; Fax: +39 50 551290; Email: l.migliore{at}geog.unipi.it
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Received on August 6, 1998; accepted on October 16, 1998.
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