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Increased perforin expression in multiple sclerosis patients during exacerbation of disease in peripheral blood lymphocytes
Journal of Neuroimmunology 74 Ž1997. 198–204
Increased perforin expression in multiple sclerosis patients during exacerbation of disease in peripheral blood lymphocytes Gordana Rubesa ˇ a, Eckhard R. Podack b, Juraj Sepcic ˇ ´ c , Daniel Rukavina a
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Department of Physiology and Immunology, Medical Faculty, UniÕersity of Rijeka, Rijeka, Croatia b Department of Microbiology and Immunology, UniÕersity of Miami, Miami, USA c Department of Neurology, Medical Faculty, UniÕersity of Rijeka, Rijeka, Croatia Received 26 December 1995; revised 18 March 1996; accepted 30 May 1996
Abstract The expression of perforin ŽP. in subpopulations of the PBL of multiple sclerosis ŽMS. patients in stable and active phase of disease was investigated, by simultaneous detection of P Žintracellular molecule. and cell surface antigens. A significant increase of CD4qPq Ž p - 0.02. and CD16qPq Ž p - 0.001., and decrease of CD56qPq Ž p - 0.05. cells in active MS was found. In active disease there is a highly significant increase Ž p - 0.001. of average fluorescence intensity ŽAFI. for P in CD4 dimq cells, and these cells are larger in size and have higher granularity Ž p - 0.05. compared to CD4 brightq P dimq cells. Surprisingly, there were no CD25q Pq cells in either group of MS patients. These results show that CD4qPq cells are upregulated in active disease in cell number, in the level of P expression per cell, and in the level of cell activation Žincrease in cell size and granularity.. It is suggested that CD4qPq cytotoxic cells may play a role in the pathogenetic mechanisms of MS. Keywords: Perforin; Multiple sclerosis; Peripheral blood lymphocytes; NK; CTL
1. Introduction Killer Žcytotoxic. lymphocytes belong to the natural killer ŽNK. cell lineage and to cytotoxic T lymphocyte ŽCTL. subpopulations. The cytoplasm of cytotoxic cells is rich in granules composed of complexes of proteoglycan in association with perforin ŽP., granzymes and possibly other molecules such as T-cell intracellular antigen ŽTIA.. Perforin Žpore forming protein. is a cytolytic molecule that is a mediator in cell-mediated cytotoxicity reactions Žfor review see: Podack, 1993, 1995.. Cytotoxicity in circulating non-activated lymphocytes is restricted to NK cells and to grd TCRq T cells. P is constitutively expressed in CD3yCD56qCD16q NK cells and CD3qCD4yCD8y grd TCRqT cells, and in a subpopulation of CD8q cells simultaneously CD11bq ŽNakata et al., 1990.. The expression of P in T cells has been a marker for functionally activated cytotoxic cells in situ ŽPodack, 1991; Griffiths and Mueller, 1991.. P expression
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can be induced in many CD4q, MHC class II restricted CTL, and in CD8q CTL ŽYagita et al., 1992.. Very recently, it has also been shown that TCR abqCD4yCD8y T cells can express P and are cytotoxic ŽWu et al., 1994; Hammond et al., 1993.. P is absent from B cells and monocytes ŽYagita et al., 1992.. Cytolytic cells ŽCTL and NK cells. are not homogenous populations, as we have shown previously ŽRukavina et al., 1994, 1995.. CTL effectors can belong to both, CD8q Pq and CD4qPq subpopulations, attacking target cells upon prior activation through the recognition of peptides presented via class I or class II MHC antigens on the target cell, respectively. NK effectors can belong to CD56qPq and CD16qPq subpopulations, and they not need prior activation for execution of cytolytic function. Multiple sclerosis ŽMS. is thought to be an autoimmune disease characterized locally by demyelination of white matter associated with perivascular lymphocyte and macrophage infiltrations and many systemic immunological abnormalities. In peripheral blood ŽPB. and cerebrospinal fluid ŽCSF. levels of lymphocytes populations which normally regulate immune response oscillate ŽHafler and Weiner, 1989.. It is well known that in MS patients many immune abnormalities occur primarily in the periph-
0165-5728r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 6 . 0 0 2 3 6 - 6
G. Rubesa ˇ et al.r Journal of Neuroimmunology 74 (1997) 198–204
eral immune compartment ŽScolozzi et al., 1992. indicating that connections exist between the brain and centers of systemic immune response and that CNS antigen presentation events could take place peripherally ŽCserr and Knopf, 1992.. The role of effector T cells, both cytotoxic T lymphocytes and T helper cells, in the pathogenesis of tissue damage is still far from being completely understood ŽSatoh et al., 1991; Scolozzi et al., 1992.. The T lymphocytes found in early MS lesions are predominantly CD4q, whereas CD8q T cells become much more numerous at a later stage Žfor review see: Tuohy et al., 1994; Fabry et al., 1994.. Both, the percentage and absolute cell counts of NK, as well as their activity, were found to be significantly lower in MS patients, than in normals ŽVranesˇ et al., 1989.. In the present study we have investigated the intracellular expression of the cytolytic molecule P during the stable and active phase of MS. We have examined P expression in all subpopulations including cytotoxic T-cells and NK-cells and their subpopulations in order to directly address the question as to whether a specific cell population containing P in their granules ŽCD4q Pq, CD8q Pq, CD56q Pq, CD16q Pq . is operative in the disease process in vivo. This approach has also allowed us to address the question as to whether the different clinical courses of disease Žactive versus stable. are accompanied with different P profiles, and whether P expression could be correlated with the disease activity. The results obtained suggest pathogenetic relevance of P in this autoimmune disease.
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Patients belonging to the stable disease Ž6 patients, 3 females and 3 males. were for at least six months Žmean " SD s 41.3 " 21.9 months. without any signs of active disease. The age ranged from 35 to 55 years Žmean age of 47 years., the Kurtzke score was 5.5 " 1.2, and disease duration was 17.3 " 8.2 years. Patients with active disease Ž17 females and 4 males. had an acute attack 1.08 " 0.62 months before investigation Ž p - 0.001 versus stable disease.. The age of patients ranged from 17 to 55 years Žmean age 29.7 years., the Kurtzke score was 4.1 " 1.5 Ž p - 0.05 versus stable disease., and disease duration was 9.2 " 6.9 years Ž p - 0.03 versus stable disease.. Eleven healthy persons, consisting of healthy laboratory personnel, eight females and three males, corresponding by age to the examined patient-group’s age from 25 to 50 years were also studied. The investigations were approved by the Ethics Committee of the Medical Faculty, University of Rijeka. 2.2. Isolation of peripheral blood lymphocytes Approximately 10 ml of venous peripheral blood was drawn in a heparinized syringe, stratified over 10 ml of Ficoll-Hypaque and centrifuged for 20 min at 800 = g. Peripheral blood lymphocytes ŽPBL. were recovered, washed in RPMI 1640 and resuspended at a final concentration of 1 = 10 6 PBLrper sample in FACS buffer. Cell viability was checked by trypan blue. 2.3. Simultaneous detection of cell surface and intracellular antigens by flow cytometry
2. Materials and methods 2.1. Patients A total of 27 MS patients from the Neurology Clinic of the Clinical Hospital in Rijeka, were investigated. In accord with criteria of Poser et al. Ž1983. all patients had clinically definite and laboratory-supported definite MS. The degree of disability was determined according to the disability status scale in multiple sclerosis ŽKurtzke, 1961, 1983.. All patients have been tested for evoked potentials and intra blood–brain barrier IgG synthesis rates and CSF oligoclonal bands. In all patients magnetic resonance imaging scans for demyelinating disease were positive. In patients with stable disease control analysis showed neither the presence of new lesions nor the enlargement of the previous. In patients with active disease both were confirmed. None of the patients were taking anti-inflammatory medications for at least one month before testing and immunosuppressive treatment one year before testing. According to the course of the disease all patients belonged to remittent-progressive form. In regard to a phase of the disease all MS patients were subdivided in two groups: Ža. active disease, and Žb. stable disease.
Our method for simultaneous detection of perforin Žintracellular antigen. and cell surface antigens was described elsewhere ŽRukavina et al., 1995.. Therefore we will present herewith only the main steps. PBL were cultured overnight at 378C in complete RPMI 1640 medium containing 10% fetal calf serum ŽFCS., 2 mM L-glutamine, 100 IUrml penicillin, 100 m grml streptomycin and 5 = 10y5 M 2-mercaptoethanol. Nonadherent cells were washed twice in FACS buffer Ž2% FCS, 1 mM EDTA and 0.1% sodium azide in PBS.. Approximately 1 = 10 6 cells were fixed in 100 m l PBS containing 4% paraformaldehyde ŽpH 7.4. for 10 min at room temperature. After two washes in FACS buffer the cells were permeabilized with saponin buffer Ž0.1% saponin, Sigma; 2% goat serum and 1 mM EGTA in PBS. for 20 min at room temperature. Murine monoclonal antibody d G9 ŽIgG2b, purified from Balbrc ascites, 3 m gr100 m l. reacting with human perforin ŽHameed et al., 1992. was added to the cell suspension in saponin at a final concentration of 5–10 m g per sample and incubated for 30 min at room temperature. All further steps Žsecondary antibody — fluorescein — conjugated goat antimouse IgG, Becton Dickinson. and rinsing steps were performed in saponin buffer. All mono-
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Fig. 1. The percentage of total P-positive cells and the frequency Žpercentage of positive cellsr10000 counted. of double-positive cells Žperforinq and surface markerq . in PBL of MS patients Žwhite column, healthy control; filled column, active MS; plaid column, stable MS.. Results are expressed as mean percentage"SE. Levels of significance: ) p- 0.05; ) ) p- 0.02; ) ) ) p- 0.001.
clonal antibodies used were Becton Dickinson. Cell surface antigens were stained with phycoerythrin conjugated monoclonal antibodies: CD4 ŽLeu-3a., CD8 ŽLeu-2a., CD25 ŽAnti-IL-2R. and CD56 ŽLeu-19., or unconjugated monoclonal antibodies CD3 ŽLeu-4. and CD16 ŽLeu-11b.. When unconjugated antibodies were used the binding was visualized by using biotynilated anti-mouse Ig and streptavidin–phycoerythrin. In some experiments only surface labeling was performed. A minimum of 10 4 cells were analyzed on FACScan ŽBecton Dickinson. and the results were recorded on a floppy disk. FACS analyses were performed by a person without knowledge of the patient’s clinical status. Results are presented as relative fluorescence intensity of gated population of cells. 2.4. Statistical analysis Statistical analysis was performed using a standard Student’s t-test.
3. Results 3.1. Cell surface antigenic phenotype of PBL in MS patients (single staining) Flow cytometric analysis of PBL from MS patients in active and stable disease Žsingle staining. were performed in order to determine whether any difference in prevalence of cell phenotypes existed. Considerable differences between the group of active and stable MS and controls were found Žresults not shown.. In the active disease group a
Fig. 2. Phenotype characteristics Ždouble color flow cytometry. of gated population of perforin positive peripheral blood lymphocytes of MS patients. Results are expressed as mean percentage"SE. White column, healthy control; filled column, active MS; plaid column, stable MS. Levels of significance: ) p- 0.02; ) ) p- 0.01; ) ) ) p- 0.0001.
significant increase of the percentage of CD3q Ž p - 0.05., CD4q Ž p - 0.01., CD16q and CD25q Ž p - 0.001. cells was found accompanied by a decrease of CD56q cells Ž p - 0.01. when compared to healthy control subjects. Similar changes, i.e. significant increase of CD3q and CD4q Ž p - 0.05. as well as CD16q and CD25q Ž p 0.001. cells were found also in the active versus the stable disease group. There was no significant difference between stable MS and healthy controls. 3.2. Total P q cells and cell surface antigen expression of P q cells First we investigated the percentage of lymphocytes containing the cytolytic protein P in their granules. The percentage of total Pq cells in both MS groups was lower than in controls, but the difference was not significant ŽFig. 1.. Next PBL were double-labeled simultaneously for the intracellular antigen, perforin, and for a cell membrane surface antigen. Double staining showed similar relationships between active MS versus stable MS or healthy control group, respectively, similar to the results for single staining ŽFig. 1.. There was a marked increase of CD4q Pq and CD16qPq cells in patients with active disease while the percentage of CD56qPq cells was lower in this group compared to the healthy control or stable disease group. It is interesting to note that there were no CD25qPq cells in either group of MS patients. Determination of the phenotype of gated, Pq cells ŽFig. 2. probes the important question of the distribution of surface markers among perforinq cells in MS patients.
Fig. 3. Two color analysis of PBL for perforin Žhorizontal. and cell surface phenotype Žvertical. in MS patients with active and stable disease. ŽA. Subpopulations of CTL ŽCD4q Pq and CD8q Pq .; ŽB. subpopulations of NK cells ŽCD56q Pq and CD16q Pq ..
G. Rubesa ˇ et al.r Journal of Neuroimmunology 74 (1997) 198–204
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Table 1 Percentages of positive cells in both brightq and dimq double positive CTL ŽPq CD4q and Pq CD8q . and NK peripheral blood lymphocytes ŽPq CD56q and Pq CD16q . in stable and active multiple sclerosis Phenotype
Stable
n
Active
n
P bright CD4 bright P bright CD4 dim P dim CD4 bright P dim CD4 dim
0.00% a 0.00% 1.50% 0.00%
6 6 6 6
0.00% 2.10% 3.80% 0.00%
21 21 21 21
P bright CD8 bright P bright CD8 dim P dim CD8 bright P dim CD8 dim
0.00% 7.00% 4.80% 0.50%
6 6 6 6
0.00% 0.40% 4.85% 5.60%
21 21 21 21
14.20% 0.70%
6 6
11.80% 7.50%
21 18
Pq CD56q Pq CD16q a
Percentage of positive cellsr10000 cells.
The frequencies of CD3, CD4 and CD16 positive cells in the gated population of Pq cells in active disease were significantly higher than in both stable disease and in the control group. Again, there were no CD25q cells among Pq cells in MS patients. The investigations of P representation in the cells of various phenotypes Ždata not presented. showed a significant increase of Pq cells in the subpopulation of CD4q cells in active disease compared to both stable and control groups Ž p - 0.02.. 3.3. Changes in the leÕels of perforin and cell surface markers expression It has been shown previously that the average fluorescence intensity ŽAFI. obtained by flow cytometry reflects the relative number of antigen molecules present inron the cells ŽSumner et al., 1991.. By applying standard parameters for FSC, SSC, FL1 and FL2 we have analyzed AFI of gated double positive PBL in both MS groups. As shown in Fig. 1 there was a significant increase of CD4qPq cells in active disease compared to stable disease Žfrom 1.5% to 6%.. According to the fluorescence intensity for the CD4 molecule, lymphocytes could be divided in two subsets: CD4 brightq and CD4 dimq ŽFig. 3A and Table 1.. All CD4q cells in stable disease are P dimq, CD4 brightq. However, in active disease there is highly significant increase Ž p 0.001. of the AFI for P in CD4 dimq, i.e. CD4 dimq lymphocytes are P brightq Ž2.1% of PBL. while those expressing CD4 brightq remain P dimq Ž3.8% of PBL.. Simultaneously, CD4 dimq, P brightq cells are larger in size Žaverage fluorescence intensity: AFI 33.0 " 2.9 versus 27.7 " 1.6; p 0.05. and have higher granularity ŽAFI 115.1 " 3.2 versus 107.3 " 2.5; p - 0.05. compared to CD4 brightq P dimq cells in this group. Among CD8 cells in stable disease ŽFig. 3A; Table 1. CD8 dimq cells were largely P brightq Ž7% of PBL. and CD8 brightq were P dimq Ž4.8%.. In active disease an in-
crease Ž5.6%. of P dimq was found in the subpopulation of CD8 dimq cells. In addition a significant increase of CD16q Pq cells Ž p - 0.001. was found in active MS ŽFig. 3B; Table 1.. The average fluorescence intensity ŽAFI. for perforin was highest in CD56q cells.
4. Discussion An increased interest for the investigation of cytotoxicity and the role of P in allotransplantation, reproduction, tumor immunity and viral infections Žfor review see: Griffiths and Mueller, 1991; Rukavina et al., 1994. stems from the realization that cytotoxic lymphocytes represent an important effector arm of the immune system. This is evident from the finding that TH1 cytokines drive mostly cellular, cytotoxic responses that rely heavily on the expression and action of perforin and other granule associated proteins. In human autoimmune diseases P was found in lymphocytes obtained from the synovial fluid of patients with rheumatoid arthritis ŽYoung et al., 1992; Griffiths et al., 1992. and in thyroid-infiltrating lymphocytes ŽWu et al., 1994.. However, according to the search of the literature the role of Pq cells in MS so far has not been investigated. In the experiments presented here we have noticed significant differences in some lymphocyte subpopulations between active and stable disease. Various changes of lymphocyte subpopulations in remittingrprogressive MS patients, particularly during the acute phase of the disease, have been reported previously by others as well ŽRose et al., 1988; Gambi et al., 1991.. A highly significant increase of the level of IL-2R a ŽCD25. expressing cells, that we have observed in the acute phase of the disease, supports the hypothesis of a continuous activation of some lymphocyte subsets in MS patients ŽGambi et al., 1991.. An increased level of IL-2R expressing cells was found in MS attacks and in chronic progressive MS by others as well ŽSelmaj et al., 1986; Gallo et al., 1989.. Surprisingly, in the experiments described here, a dichotomy between IL-2R a ŽCD25. expression and P expression was found. Essentially, none of the P positive cells stain with antibody to IL-2R a , even though it is known that P-containing T-cells are also activated T cells ŽPodack, 1991.. The most interesting result arising from these experiments relates to the role that P could play in the pathogenesis of MS, particularly in exacerbation of disease. The results clearly show that CD4qPq cells are upregulated in active disease, in frequency, in the level of P expression per cell, and in the level of cell activation Žincrease in cell size and granularity.. These parameters correlated with the severity of clinical symptoms of the disease Žactive versus stable.. This finding strongly supports the hypothesis about the potential role of CD4q cytolytic effector cells in the inductionrexacerbation of MS and EAE ŽFabry et al., 1994; McCarron et al., 1991.. CD4q Pq cells are the
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subpopulation of CD4q T lymphocytes with cytotoxic potential and rapid granule mediated killing of target cells bearing MHC class II molecules and the appropriate antigen. It has been shown that most human CD4q CTL specific for HIV are Pq ŽPodack et al., 1992., as well as some of thyroid-infiltrating lymphocytes in Hashimoto’s thyreoiditis ŽWu et al., 1994.. The significance of the finding that CD4 dimq cells are P brightq in active disease is not understood. Similar observations were made in autoimmune thyroiditis ŽWu et al., 1994. suggesting a special role of this subpopulation in the disease process. The CNS has been considered to be an inducible, rather than constitutive, site for the expression of MHC molecules. Under pathological conditions, such as MS and EAE, MHC class II molecules could be expressed on various kinds of cells including cerebral microvessel endothelial cells, and they could become antigen presenting cells Žfor review see: Fabry et al., 1994.. Further, MHC class II molecules may also participate in the passage of cells through the blood–brain barrier. It has been proposed that antigen specific lysis of cerebral endothelial cells by MHC class II restricted cytotoxic lymphocytes could be an important mechanism for the migration of lymphocytes into the brain parenchyma ŽMcCarron et al., 1991.. CD4q Pq subpopulations of peripheral blood lymphocytes, that significantly increase in the active phase of MS, could be the candidate population of cytotoxic cells for this function. Strong increases of CD4q cells among P-containing PBL Ž18% in active versus 4% in stable disease. indicate the potential for destruction of cells presenting MHC class II in association with specific antigen. In experiments in vitro it has been shown that oligodendrocytes, which synthesize and maintain myelin in the CNS, are highly susceptible to attack by T cell perforin ŽScolding et al., 1990.. Therefore, in the case of blood–brain barrier disruption, attack by P-containing cells may cause transient or irreversible myelin injury. Interestingly, there were no significant changes for the CD8q Pq subpopulations Žsee Fig. 1., having the potential of killing target cells bearing class I MHC molecules and appropriate antigen. Mechanisms of regulation of P expression by cytolytic cells are of both fundamental and clinical interest. Based on differences in the level of P expression ŽAFI., CD4q Pq cells in active disease belong to four subpopulations: P brightq and P dimq, CD4 brightq and CD4 dimq ŽTable 1.. It is clear that P expression in T-cells is a sign of functional activation ŽPodack, 1991; Griffiths and Mueller, 1991.. In active disease it appears that only a small part of CD4qPq cells are activated ŽP brightq .. The observation of Held et al. Ž1993. that after antigen specific activation in vitro a small fraction of the CD4q cell line Q4, used for adoptive transfer of EAE in rats, expressed the P gene, is in the line with this assumption. A role for lymphocyte mediated antibody dependent cellular cytotoxicity ŽADCC. in disease activation is suggested by significant increase of CD16qPq cells, and
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particularly by a seven fold increase of CD16q cells among total Pq cells. The CD16 IgGFcg RIIIA receptor is a conventional transmembrane protein, with a distinct cytoplasmic domain ŽRavetch and Kinet, 1991.. Fcg RIIIA is expressed on NK cells ŽRavetch and Perussia, 1989., and is exclusively responsible for ADCC. The CD16 - NK subset lacks this function ŽNagler et al., 1989.. Therefore, significant increases of CD16qPq cells in the active phase of MS could point to a role for ADCC as a mechanism of tissue damage.
Acknowledgements The results presented in this investigation are financially supported by the Ministry of Science and Technology of the Republic of Croatia and by NIH grant CA 392012. We are grateful to Mrs. Ivana Godnic´ for skillfully typing the manuscript and to Mrs. Davorka Percinic ˇ ´ for excellent technical assistance.
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Increased perforin expression in multiple sclerosis patients during exacerbation of disease in peripheral blood lymphocytes Gordana Rubesa ˇ a, Eckhard R. Podack b, Juraj Sepcic ˇ ´ c , Daniel Rukavina a
a,)
Department of Physiology and Immunology, Medical Faculty, UniÕersity of Rijeka, Rijeka, Croatia b Department of Microbiology and Immunology, UniÕersity of Miami, Miami, USA c Department of Neurology, Medical Faculty, UniÕersity of Rijeka, Rijeka, Croatia Received 26 December 1995; revised 18 March 1996; accepted 30 May 1996
Abstract The expression of perforin ŽP. in subpopulations of the PBL of multiple sclerosis ŽMS. patients in stable and active phase of disease was investigated, by simultaneous detection of P Žintracellular molecule. and cell surface antigens. A significant increase of CD4qPq Ž p - 0.02. and CD16qPq Ž p - 0.001., and decrease of CD56qPq Ž p - 0.05. cells in active MS was found. In active disease there is a highly significant increase Ž p - 0.001. of average fluorescence intensity ŽAFI. for P in CD4 dimq cells, and these cells are larger in size and have higher granularity Ž p - 0.05. compared to CD4 brightq P dimq cells. Surprisingly, there were no CD25q Pq cells in either group of MS patients. These results show that CD4qPq cells are upregulated in active disease in cell number, in the level of P expression per cell, and in the level of cell activation Žincrease in cell size and granularity.. It is suggested that CD4qPq cytotoxic cells may play a role in the pathogenetic mechanisms of MS. Keywords: Perforin; Multiple sclerosis; Peripheral blood lymphocytes; NK; CTL
1. Introduction Killer Žcytotoxic. lymphocytes belong to the natural killer ŽNK. cell lineage and to cytotoxic T lymphocyte ŽCTL. subpopulations. The cytoplasm of cytotoxic cells is rich in granules composed of complexes of proteoglycan in association with perforin ŽP., granzymes and possibly other molecules such as T-cell intracellular antigen ŽTIA.. Perforin Žpore forming protein. is a cytolytic molecule that is a mediator in cell-mediated cytotoxicity reactions Žfor review see: Podack, 1993, 1995.. Cytotoxicity in circulating non-activated lymphocytes is restricted to NK cells and to grd TCRq T cells. P is constitutively expressed in CD3yCD56qCD16q NK cells and CD3qCD4yCD8y grd TCRqT cells, and in a subpopulation of CD8q cells simultaneously CD11bq ŽNakata et al., 1990.. The expression of P in T cells has been a marker for functionally activated cytotoxic cells in situ ŽPodack, 1991; Griffiths and Mueller, 1991.. P expression
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can be induced in many CD4q, MHC class II restricted CTL, and in CD8q CTL ŽYagita et al., 1992.. Very recently, it has also been shown that TCR abqCD4yCD8y T cells can express P and are cytotoxic ŽWu et al., 1994; Hammond et al., 1993.. P is absent from B cells and monocytes ŽYagita et al., 1992.. Cytolytic cells ŽCTL and NK cells. are not homogenous populations, as we have shown previously ŽRukavina et al., 1994, 1995.. CTL effectors can belong to both, CD8q Pq and CD4qPq subpopulations, attacking target cells upon prior activation through the recognition of peptides presented via class I or class II MHC antigens on the target cell, respectively. NK effectors can belong to CD56qPq and CD16qPq subpopulations, and they not need prior activation for execution of cytolytic function. Multiple sclerosis ŽMS. is thought to be an autoimmune disease characterized locally by demyelination of white matter associated with perivascular lymphocyte and macrophage infiltrations and many systemic immunological abnormalities. In peripheral blood ŽPB. and cerebrospinal fluid ŽCSF. levels of lymphocytes populations which normally regulate immune response oscillate ŽHafler and Weiner, 1989.. It is well known that in MS patients many immune abnormalities occur primarily in the periph-
0165-5728r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 6 . 0 0 2 3 6 - 6
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eral immune compartment ŽScolozzi et al., 1992. indicating that connections exist between the brain and centers of systemic immune response and that CNS antigen presentation events could take place peripherally ŽCserr and Knopf, 1992.. The role of effector T cells, both cytotoxic T lymphocytes and T helper cells, in the pathogenesis of tissue damage is still far from being completely understood ŽSatoh et al., 1991; Scolozzi et al., 1992.. The T lymphocytes found in early MS lesions are predominantly CD4q, whereas CD8q T cells become much more numerous at a later stage Žfor review see: Tuohy et al., 1994; Fabry et al., 1994.. Both, the percentage and absolute cell counts of NK, as well as their activity, were found to be significantly lower in MS patients, than in normals ŽVranesˇ et al., 1989.. In the present study we have investigated the intracellular expression of the cytolytic molecule P during the stable and active phase of MS. We have examined P expression in all subpopulations including cytotoxic T-cells and NK-cells and their subpopulations in order to directly address the question as to whether a specific cell population containing P in their granules ŽCD4q Pq, CD8q Pq, CD56q Pq, CD16q Pq . is operative in the disease process in vivo. This approach has also allowed us to address the question as to whether the different clinical courses of disease Žactive versus stable. are accompanied with different P profiles, and whether P expression could be correlated with the disease activity. The results obtained suggest pathogenetic relevance of P in this autoimmune disease.
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Patients belonging to the stable disease Ž6 patients, 3 females and 3 males. were for at least six months Žmean " SD s 41.3 " 21.9 months. without any signs of active disease. The age ranged from 35 to 55 years Žmean age of 47 years., the Kurtzke score was 5.5 " 1.2, and disease duration was 17.3 " 8.2 years. Patients with active disease Ž17 females and 4 males. had an acute attack 1.08 " 0.62 months before investigation Ž p - 0.001 versus stable disease.. The age of patients ranged from 17 to 55 years Žmean age 29.7 years., the Kurtzke score was 4.1 " 1.5 Ž p - 0.05 versus stable disease., and disease duration was 9.2 " 6.9 years Ž p - 0.03 versus stable disease.. Eleven healthy persons, consisting of healthy laboratory personnel, eight females and three males, corresponding by age to the examined patient-group’s age from 25 to 50 years were also studied. The investigations were approved by the Ethics Committee of the Medical Faculty, University of Rijeka. 2.2. Isolation of peripheral blood lymphocytes Approximately 10 ml of venous peripheral blood was drawn in a heparinized syringe, stratified over 10 ml of Ficoll-Hypaque and centrifuged for 20 min at 800 = g. Peripheral blood lymphocytes ŽPBL. were recovered, washed in RPMI 1640 and resuspended at a final concentration of 1 = 10 6 PBLrper sample in FACS buffer. Cell viability was checked by trypan blue. 2.3. Simultaneous detection of cell surface and intracellular antigens by flow cytometry
2. Materials and methods 2.1. Patients A total of 27 MS patients from the Neurology Clinic of the Clinical Hospital in Rijeka, were investigated. In accord with criteria of Poser et al. Ž1983. all patients had clinically definite and laboratory-supported definite MS. The degree of disability was determined according to the disability status scale in multiple sclerosis ŽKurtzke, 1961, 1983.. All patients have been tested for evoked potentials and intra blood–brain barrier IgG synthesis rates and CSF oligoclonal bands. In all patients magnetic resonance imaging scans for demyelinating disease were positive. In patients with stable disease control analysis showed neither the presence of new lesions nor the enlargement of the previous. In patients with active disease both were confirmed. None of the patients were taking anti-inflammatory medications for at least one month before testing and immunosuppressive treatment one year before testing. According to the course of the disease all patients belonged to remittent-progressive form. In regard to a phase of the disease all MS patients were subdivided in two groups: Ža. active disease, and Žb. stable disease.
Our method for simultaneous detection of perforin Žintracellular antigen. and cell surface antigens was described elsewhere ŽRukavina et al., 1995.. Therefore we will present herewith only the main steps. PBL were cultured overnight at 378C in complete RPMI 1640 medium containing 10% fetal calf serum ŽFCS., 2 mM L-glutamine, 100 IUrml penicillin, 100 m grml streptomycin and 5 = 10y5 M 2-mercaptoethanol. Nonadherent cells were washed twice in FACS buffer Ž2% FCS, 1 mM EDTA and 0.1% sodium azide in PBS.. Approximately 1 = 10 6 cells were fixed in 100 m l PBS containing 4% paraformaldehyde ŽpH 7.4. for 10 min at room temperature. After two washes in FACS buffer the cells were permeabilized with saponin buffer Ž0.1% saponin, Sigma; 2% goat serum and 1 mM EGTA in PBS. for 20 min at room temperature. Murine monoclonal antibody d G9 ŽIgG2b, purified from Balbrc ascites, 3 m gr100 m l. reacting with human perforin ŽHameed et al., 1992. was added to the cell suspension in saponin at a final concentration of 5–10 m g per sample and incubated for 30 min at room temperature. All further steps Žsecondary antibody — fluorescein — conjugated goat antimouse IgG, Becton Dickinson. and rinsing steps were performed in saponin buffer. All mono-
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Fig. 1. The percentage of total P-positive cells and the frequency Žpercentage of positive cellsr10000 counted. of double-positive cells Žperforinq and surface markerq . in PBL of MS patients Žwhite column, healthy control; filled column, active MS; plaid column, stable MS.. Results are expressed as mean percentage"SE. Levels of significance: ) p- 0.05; ) ) p- 0.02; ) ) ) p- 0.001.
clonal antibodies used were Becton Dickinson. Cell surface antigens were stained with phycoerythrin conjugated monoclonal antibodies: CD4 ŽLeu-3a., CD8 ŽLeu-2a., CD25 ŽAnti-IL-2R. and CD56 ŽLeu-19., or unconjugated monoclonal antibodies CD3 ŽLeu-4. and CD16 ŽLeu-11b.. When unconjugated antibodies were used the binding was visualized by using biotynilated anti-mouse Ig and streptavidin–phycoerythrin. In some experiments only surface labeling was performed. A minimum of 10 4 cells were analyzed on FACScan ŽBecton Dickinson. and the results were recorded on a floppy disk. FACS analyses were performed by a person without knowledge of the patient’s clinical status. Results are presented as relative fluorescence intensity of gated population of cells. 2.4. Statistical analysis Statistical analysis was performed using a standard Student’s t-test.
3. Results 3.1. Cell surface antigenic phenotype of PBL in MS patients (single staining) Flow cytometric analysis of PBL from MS patients in active and stable disease Žsingle staining. were performed in order to determine whether any difference in prevalence of cell phenotypes existed. Considerable differences between the group of active and stable MS and controls were found Žresults not shown.. In the active disease group a
Fig. 2. Phenotype characteristics Ždouble color flow cytometry. of gated population of perforin positive peripheral blood lymphocytes of MS patients. Results are expressed as mean percentage"SE. White column, healthy control; filled column, active MS; plaid column, stable MS. Levels of significance: ) p- 0.02; ) ) p- 0.01; ) ) ) p- 0.0001.
significant increase of the percentage of CD3q Ž p - 0.05., CD4q Ž p - 0.01., CD16q and CD25q Ž p - 0.001. cells was found accompanied by a decrease of CD56q cells Ž p - 0.01. when compared to healthy control subjects. Similar changes, i.e. significant increase of CD3q and CD4q Ž p - 0.05. as well as CD16q and CD25q Ž p 0.001. cells were found also in the active versus the stable disease group. There was no significant difference between stable MS and healthy controls. 3.2. Total P q cells and cell surface antigen expression of P q cells First we investigated the percentage of lymphocytes containing the cytolytic protein P in their granules. The percentage of total Pq cells in both MS groups was lower than in controls, but the difference was not significant ŽFig. 1.. Next PBL were double-labeled simultaneously for the intracellular antigen, perforin, and for a cell membrane surface antigen. Double staining showed similar relationships between active MS versus stable MS or healthy control group, respectively, similar to the results for single staining ŽFig. 1.. There was a marked increase of CD4q Pq and CD16qPq cells in patients with active disease while the percentage of CD56qPq cells was lower in this group compared to the healthy control or stable disease group. It is interesting to note that there were no CD25qPq cells in either group of MS patients. Determination of the phenotype of gated, Pq cells ŽFig. 2. probes the important question of the distribution of surface markers among perforinq cells in MS patients.
Fig. 3. Two color analysis of PBL for perforin Žhorizontal. and cell surface phenotype Žvertical. in MS patients with active and stable disease. ŽA. Subpopulations of CTL ŽCD4q Pq and CD8q Pq .; ŽB. subpopulations of NK cells ŽCD56q Pq and CD16q Pq ..
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Table 1 Percentages of positive cells in both brightq and dimq double positive CTL ŽPq CD4q and Pq CD8q . and NK peripheral blood lymphocytes ŽPq CD56q and Pq CD16q . in stable and active multiple sclerosis Phenotype
Stable
n
Active
n
P bright CD4 bright P bright CD4 dim P dim CD4 bright P dim CD4 dim
0.00% a 0.00% 1.50% 0.00%
6 6 6 6
0.00% 2.10% 3.80% 0.00%
21 21 21 21
P bright CD8 bright P bright CD8 dim P dim CD8 bright P dim CD8 dim
0.00% 7.00% 4.80% 0.50%
6 6 6 6
0.00% 0.40% 4.85% 5.60%
21 21 21 21
14.20% 0.70%
6 6
11.80% 7.50%
21 18
Pq CD56q Pq CD16q a
Percentage of positive cellsr10000 cells.
The frequencies of CD3, CD4 and CD16 positive cells in the gated population of Pq cells in active disease were significantly higher than in both stable disease and in the control group. Again, there were no CD25q cells among Pq cells in MS patients. The investigations of P representation in the cells of various phenotypes Ždata not presented. showed a significant increase of Pq cells in the subpopulation of CD4q cells in active disease compared to both stable and control groups Ž p - 0.02.. 3.3. Changes in the leÕels of perforin and cell surface markers expression It has been shown previously that the average fluorescence intensity ŽAFI. obtained by flow cytometry reflects the relative number of antigen molecules present inron the cells ŽSumner et al., 1991.. By applying standard parameters for FSC, SSC, FL1 and FL2 we have analyzed AFI of gated double positive PBL in both MS groups. As shown in Fig. 1 there was a significant increase of CD4qPq cells in active disease compared to stable disease Žfrom 1.5% to 6%.. According to the fluorescence intensity for the CD4 molecule, lymphocytes could be divided in two subsets: CD4 brightq and CD4 dimq ŽFig. 3A and Table 1.. All CD4q cells in stable disease are P dimq, CD4 brightq. However, in active disease there is highly significant increase Ž p 0.001. of the AFI for P in CD4 dimq, i.e. CD4 dimq lymphocytes are P brightq Ž2.1% of PBL. while those expressing CD4 brightq remain P dimq Ž3.8% of PBL.. Simultaneously, CD4 dimq, P brightq cells are larger in size Žaverage fluorescence intensity: AFI 33.0 " 2.9 versus 27.7 " 1.6; p 0.05. and have higher granularity ŽAFI 115.1 " 3.2 versus 107.3 " 2.5; p - 0.05. compared to CD4 brightq P dimq cells in this group. Among CD8 cells in stable disease ŽFig. 3A; Table 1. CD8 dimq cells were largely P brightq Ž7% of PBL. and CD8 brightq were P dimq Ž4.8%.. In active disease an in-
crease Ž5.6%. of P dimq was found in the subpopulation of CD8 dimq cells. In addition a significant increase of CD16q Pq cells Ž p - 0.001. was found in active MS ŽFig. 3B; Table 1.. The average fluorescence intensity ŽAFI. for perforin was highest in CD56q cells.
4. Discussion An increased interest for the investigation of cytotoxicity and the role of P in allotransplantation, reproduction, tumor immunity and viral infections Žfor review see: Griffiths and Mueller, 1991; Rukavina et al., 1994. stems from the realization that cytotoxic lymphocytes represent an important effector arm of the immune system. This is evident from the finding that TH1 cytokines drive mostly cellular, cytotoxic responses that rely heavily on the expression and action of perforin and other granule associated proteins. In human autoimmune diseases P was found in lymphocytes obtained from the synovial fluid of patients with rheumatoid arthritis ŽYoung et al., 1992; Griffiths et al., 1992. and in thyroid-infiltrating lymphocytes ŽWu et al., 1994.. However, according to the search of the literature the role of Pq cells in MS so far has not been investigated. In the experiments presented here we have noticed significant differences in some lymphocyte subpopulations between active and stable disease. Various changes of lymphocyte subpopulations in remittingrprogressive MS patients, particularly during the acute phase of the disease, have been reported previously by others as well ŽRose et al., 1988; Gambi et al., 1991.. A highly significant increase of the level of IL-2R a ŽCD25. expressing cells, that we have observed in the acute phase of the disease, supports the hypothesis of a continuous activation of some lymphocyte subsets in MS patients ŽGambi et al., 1991.. An increased level of IL-2R expressing cells was found in MS attacks and in chronic progressive MS by others as well ŽSelmaj et al., 1986; Gallo et al., 1989.. Surprisingly, in the experiments described here, a dichotomy between IL-2R a ŽCD25. expression and P expression was found. Essentially, none of the P positive cells stain with antibody to IL-2R a , even though it is known that P-containing T-cells are also activated T cells ŽPodack, 1991.. The most interesting result arising from these experiments relates to the role that P could play in the pathogenesis of MS, particularly in exacerbation of disease. The results clearly show that CD4qPq cells are upregulated in active disease, in frequency, in the level of P expression per cell, and in the level of cell activation Žincrease in cell size and granularity.. These parameters correlated with the severity of clinical symptoms of the disease Žactive versus stable.. This finding strongly supports the hypothesis about the potential role of CD4q cytolytic effector cells in the inductionrexacerbation of MS and EAE ŽFabry et al., 1994; McCarron et al., 1991.. CD4q Pq cells are the
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subpopulation of CD4q T lymphocytes with cytotoxic potential and rapid granule mediated killing of target cells bearing MHC class II molecules and the appropriate antigen. It has been shown that most human CD4q CTL specific for HIV are Pq ŽPodack et al., 1992., as well as some of thyroid-infiltrating lymphocytes in Hashimoto’s thyreoiditis ŽWu et al., 1994.. The significance of the finding that CD4 dimq cells are P brightq in active disease is not understood. Similar observations were made in autoimmune thyroiditis ŽWu et al., 1994. suggesting a special role of this subpopulation in the disease process. The CNS has been considered to be an inducible, rather than constitutive, site for the expression of MHC molecules. Under pathological conditions, such as MS and EAE, MHC class II molecules could be expressed on various kinds of cells including cerebral microvessel endothelial cells, and they could become antigen presenting cells Žfor review see: Fabry et al., 1994.. Further, MHC class II molecules may also participate in the passage of cells through the blood–brain barrier. It has been proposed that antigen specific lysis of cerebral endothelial cells by MHC class II restricted cytotoxic lymphocytes could be an important mechanism for the migration of lymphocytes into the brain parenchyma ŽMcCarron et al., 1991.. CD4q Pq subpopulations of peripheral blood lymphocytes, that significantly increase in the active phase of MS, could be the candidate population of cytotoxic cells for this function. Strong increases of CD4q cells among P-containing PBL Ž18% in active versus 4% in stable disease. indicate the potential for destruction of cells presenting MHC class II in association with specific antigen. In experiments in vitro it has been shown that oligodendrocytes, which synthesize and maintain myelin in the CNS, are highly susceptible to attack by T cell perforin ŽScolding et al., 1990.. Therefore, in the case of blood–brain barrier disruption, attack by P-containing cells may cause transient or irreversible myelin injury. Interestingly, there were no significant changes for the CD8q Pq subpopulations Žsee Fig. 1., having the potential of killing target cells bearing class I MHC molecules and appropriate antigen. Mechanisms of regulation of P expression by cytolytic cells are of both fundamental and clinical interest. Based on differences in the level of P expression ŽAFI., CD4q Pq cells in active disease belong to four subpopulations: P brightq and P dimq, CD4 brightq and CD4 dimq ŽTable 1.. It is clear that P expression in T-cells is a sign of functional activation ŽPodack, 1991; Griffiths and Mueller, 1991.. In active disease it appears that only a small part of CD4qPq cells are activated ŽP brightq .. The observation of Held et al. Ž1993. that after antigen specific activation in vitro a small fraction of the CD4q cell line Q4, used for adoptive transfer of EAE in rats, expressed the P gene, is in the line with this assumption. A role for lymphocyte mediated antibody dependent cellular cytotoxicity ŽADCC. in disease activation is suggested by significant increase of CD16qPq cells, and
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particularly by a seven fold increase of CD16q cells among total Pq cells. The CD16 IgGFcg RIIIA receptor is a conventional transmembrane protein, with a distinct cytoplasmic domain ŽRavetch and Kinet, 1991.. Fcg RIIIA is expressed on NK cells ŽRavetch and Perussia, 1989., and is exclusively responsible for ADCC. The CD16 - NK subset lacks this function ŽNagler et al., 1989.. Therefore, significant increases of CD16qPq cells in the active phase of MS could point to a role for ADCC as a mechanism of tissue damage.
Acknowledgements The results presented in this investigation are financially supported by the Ministry of Science and Technology of the Republic of Croatia and by NIH grant CA 392012. We are grateful to Mrs. Ivana Godnic´ for skillfully typing the manuscript and to Mrs. Davorka Percinic ˇ ´ for excellent technical assistance.
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