REPORT NONSEGMENTAL VITILIGO: DECREASE O¥ THE CD45RA+ T-CELL SUBSET AND EVIDENCE FOR PERIPHERAL T-CELL ACTIVATION M. BADAWY ABDEL NASER, M.D., WOLE-DIETER LUDWIG, M.D., HARALD GOLLNICK, M.D., AND CONSTANTIN E. OREANOS, M.D. Abstract Peripheral T-lymphocytes from 16 randomly selected pa- tients with nonsegmental vitiligo were labeled with mono- clonal antibodies recognizing T-cell receptor (TCR) a/p, TCR 7/6 , CD3, CD4, CD8, CD45RA, CD45RO, C D l i b , CD11C, CD16, CD56, CD25, CD54, and HLA-DR antigens. In comparison with matched controls, a significant decrease ofthe CD45RA+ sub- set (P< 0.03) together with significant increase of the circu- lating HLA-DR+ cells (P< 0.02) was found. No other alterations were detected. These findings may point to some autoimmune phenomena involved in the pathogene- sis of the disease. The increased HLA-DR expression indi- cates the presence of activated peripheral T-cells. Thus, our data provide new and further evidence for T-cell dysregula- tion in nonsegmental vitiligo. Vitiligo is a common pigmentary disorder, whicb af- fects not less tban 1% of tbe world's population.' The etiology of the disease is widely believed to be of au- toimmune origin. This bypotbesis is based on recent experimental findings and the frequent association of vitiligo witb autoimmune diseases, such as multiple glandular insufficiencies,^ thyroid diseases,' alopecia areata,''-^ lupus erythematosus,* pernicious anemia,^ type 1 diabetes mellitus,•* Addison's disease,' and au- toimmune hypoparathyroidism.' Various aberrations in peripheral mononuclear cells, especially T cells and T-cell subsets, bave been described not only in patients with vitiligo but also among their first-degree relatives;'""'"' however, the re- sults of tbese reports have been inconsistent (Table 1). Thus we re-evaluate the present state of these major subpopulations in a series of 16 patients witb nonseg- tnental vitiligo by using FACScan analysis under stan- dardized conditions. From the Departments of Dermatology, and Hematology and Oncology, University Medical Center Steglitz, The Free Uriiver- sity of Berlin, Berlin, Germany, and the Department of Derma- tology, Ain Shams University Hospital, Cairo, Egypt. Address for correspondence: M. Badawy Abdel-Naser, M.D., Department of Dermatology, University Medical Center Steglitz, The Free University of Berlin, Hindenburgdamm 30, 1000 Berlin 45, Germany. Material and Methods Sixteen patients (5 men and 11 women; mean age 38.3 ± 13.7) with widespread nonsegmental vitiligo were included in this study. None of them had received any systemic or topical treatment within the last 6 months before the onset of our study. The clinical data are presented in Table 2. The control group consisted of 10 healthy hospital employees (4 men and 6 women; mean age 31 ± 7). All blood samples were taken under standardized conditions, i.e., 8:30-9:00 a.m., and processed shortly thereafter; the entire analysis of all specimens was completed within 2 weeks. Lympho- cytes were separated and labeled as previously described.^^ In short, lymphocytes were obtained by centrifuging diluted (1:2 with 0.1% NaCI solution) heparinized whole blood on a Ficoll-Hypaque gradient and were recovered at the inter- face. They were then washed with PBS plus 1% albumin 3 X, and the cell number was adjusted at 4x10^/mL in 0.5 mL medium. Twenty-five microliters of diluted primary MoAbs (listed in Table 3) were placed on the surface of the pellet; the solution was mixed thoroughly and incubated at 4°C for 30 min. After incubation, the cells were washed twice and mixed thoroughly with 25 pL of goat antimouse FITC conju- gated antibody; the mixture was then incubated at 4°C for 30 min. Flow cytometry was carried out using a FACScan (Becton Dickinson). A gate was set around the lymphocyte CD3 90 -1 ^ 80 - a 1 70H ± 60 H '-40H 30 - I 1 Control Vitiligo patients Figure 1. Percent values of CD3+ cells in vitiligo patients and control individuals (P> 0.05). 321
Transcript
REPORT
NONSEGMENTAL VITILIGO: DECREASE O¥ THECD45RA+ T-CELL SUBSET AND EVIDENCE FOR
PERIPHERAL T-CELL ACTIVATION
M. BADAWY ABDEL NASER, M.D., WOLE-DIETER LUDWIG, M.D.,
HARALD GOLLNICK, M.D., AND CONSTANTIN E. OREANOS, M.D.
AbstractPeripheral T-lymphocytes from 16 randomly selected pa-tients with nonsegmental vitiligo were labeled with mono-clonal antibodies recognizing T-cell receptor (TCR) a/p, TCR7/6 , CD3, CD4, CD8, CD45RA, CD45RO, CDl ib , CD11C, CD16, CD56,
CD25, CD54, and HLA-DR antigens. In comparison withmatched controls, a significant decrease ofthe CD45RA+ sub-set (P< 0.03) together with significant increase of the circu-lating HLA-DR+ cells (P< 0.02) was found. No otheralterations were detected. These findings may point tosome autoimmune phenomena involved in the pathogene-sis of the disease. The increased HLA-DR expression indi-cates the presence of activated peripheral T-cells. Thus, ourdata provide new and further evidence for T-cell dysregula-tion in nonsegmental vitiligo.
Vitiligo is a common pigmentary disorder, whicb af-fects not less tban 1% of tbe world's population.' Theetiology of the disease is widely believed to be of au-toimmune origin. This bypotbesis is based on recentexperimental findings and the frequent association ofvitiligo witb autoimmune diseases, such as multipleglandular insufficiencies,^ thyroid diseases,' alopeciaareata,''-^ lupus erythematosus,* pernicious anemia,^type 1 diabetes mellitus,•* Addison's disease,' and au-toimmune hypoparathyroidism.'
Various aberrations in peripheral mononuclearcells, especially T cells and T-cell subsets, bave beendescribed not only in patients with vitiligo but alsoamong their first-degree relatives;'""'"' however, the re-sults of tbese reports have been inconsistent (Table 1).Thus we re-evaluate the present state of these majorsubpopulations in a series of 16 patients witb nonseg-tnental vitiligo by using FACScan analysis under stan-dardized conditions.
From the Departments of Dermatology, and Hematology andOncology, University Medical Center Steglitz, The Free Uriiver-sity of Berlin, Berlin, Germany, and the Department of Derma-tology, Ain Shams University Hospital, Cairo, Egypt.
Address for correspondence: M. Badawy Abdel-Naser, M.D.,Department of Dermatology, University Medical CenterSteglitz, The Free University of Berlin, Hindenburgdamm 30,1000 Berlin 45, Germany.
Material and MethodsSixteen patients (5 men and 11 women; mean age 38.3 ±13.7) with widespread nonsegmental vitiligo were includedin this study. None of them had received any systemic ortopical treatment within the last 6 months before the onsetof our study. The clinical data are presented in Table 2. Thecontrol group consisted of 10 healthy hospital employees (4men and 6 women; mean age 31 ± 7). All blood sampleswere taken under standardized conditions, i.e., 8:30-9:00a.m., and processed shortly thereafter; the entire analysisof all specimens was completed within 2 weeks. Lympho-cytes were separated and labeled as previously described.^^In short, lymphocytes were obtained by centrifuging diluted(1:2 with 0.1% NaCI solution) heparinized whole blood on aFicoll-Hypaque gradient and were recovered at the inter-face. They were then washed with PBS plus 1% albumin 3 X,and the cell number was adjusted at 4x10^/mL in 0.5 mLmedium. Twenty-five microliters of diluted primary MoAbs(listed in Table 3) were placed on the surface of the pellet;the solution was mixed thoroughly and incubated at 4°C for30 min. After incubation, the cells were washed twice andmixed thoroughly with 25 pL of goat antimouse FITC conju-gated antibody; the mixture was then incubated at 4°C for30 min. Flow cytometry was carried out using a FACScan(Becton Dickinson). A gate was set around the lymphocyte
CD390 -1
^ 80 -a
1 70H
± 60 H
' -40H
30 - I 1Control Vitiligo
patients
Figure 1. Percent values of CD3+ cells in vitiligo patientsand control individuals (P> 0.05).
321
International Journal of DermatologyVol, 31, No, 5, May 1992
Table 1. Peripheral Blood T Cell and T-Cell Subpopulations in Vitiligo
Author CD,!+ CD4+ NK Cells CD4+/CD8-t
RatioNo. ofPatients
Soubiran"Halder'2
Grimes'^
Ghoneum'"
Mozzanica"
Mozzanica'^
D'Amelio'^
Baumer'"
Abdel-Naser'-*
NormalDecreased
Normal—
—
—
Normal
Normal
Normal
Increased
Decreased
Decreased
Decreased
Increased
Normal
Normal
Increased
Increased
Normal
,
Decreased
Normal
Normal
Normal
—
IncreasedLeu 7+ cells
—
Decreasedfunction
Increasedfunction
—
—
Normal
Normal
Increased
Decreased
Decreased
—
Increased
Normal
Normal
32
25
20
18
10
12
22
25
16
'This paper.
Table 2. Clinical Data According to Vitiligo Patients' Statements
region to exclude monocytes and dead lymphocytes fromthe estimation." The total number of fluorescent cells wasexpressed as a percentage of the total number of lympho-cytes. The percentages of T-lymphocytes and their subsetswere statistically compared to the controls by using theMann-Whitney U-Wilcoxon rank sum W-test.
RESULTS
The results are presented in Table 4 and Figures 1 to 7.The upper and lower bars represent 75% and 25%values of the positively labeled cells respectively. Themiddle bar represents the median value. As shown, asignificant decrease (P < 0.03) of CD4,5RA+ T-lympho-cytes was detected. There was no significant alteration(P > 0.05) of the TCR a/p, TCR y5, CD3+, and CD45RO+fractions or the CD4+ and CD8+ subsets.
Measurement of the activation antigens revealed asignificant increase (P < 0,02) of HLA-DR+ and some in-crease (P> 0.05) of CD25+ and CD54+ cell populations.In contrast, the values for CD16+ and CD56+ MoAbs de-tecting natural killer (NK) ceils were unchanged. Fur-thermore, no significant change of CDllb+ and CD11C+cell populations was detected (P > 0.05).
DISCUSSION
Examination of peripheral blood T-lymphocytes (PBTL)
in a series of 16 patients with nonsegmental vitiligoshowed a significant decrease of circulating CD45RA+and a significant increase of HLA-DR+ cells. The TCR
a/(3, TCR Y/5, CD3, CD4, CD8, CD45RO, CDllb, CDllC, CD16,CD56, CD25, and CD54 cell populations were all within
322
NonsegnientLil VitiligoAbdcl-Naser et al.
TCR9 0 •
I 80
^ 70
3 60
LoO
30
Control Vitiligopatients
2 10
TCR 7/6
Control Vitiligopatients
Figure 2. Percent values of TCR a/p+ and TCR Y/5+ cells invitiligo patients and control individuals (P > 0.05).
normal limits. Tbese findings are in agreement witbthose recently reported by Baumer et al.'" In contrast,otber investigators bave demonstrated cbanges of PBTLin vitiligo (see Table 1). Despite tbeir contradictory re-sults, aberrations of PBTL were regarded as direct evi-dence for involvement of tbe imtnune systetn in vitiligo.
Peripheral blood T-lympbocytes can be influencedby several conditions, sucb as stress, smoking, drugs,sport activities, and aging.^' Moreover, tbe PBTL sub-populations vary markedly according to the samplingtime (i.e., circadian rhythm) and evaluation tech-niques.^- Several explanations could, therefore, be putforward to explain tbe contradictions betweeti the re-
Table 3. List of Primary MoAbs UsedAntigen MoAB Reactivity'' Source Dilution
TCR a / p
TCR 7/5
CD3
CD4
CD45 RA
CD45 RO
CDS
CDllh
CDllC
CD16
CD56
CD54
CD25
HLA-DR
WT3I
TCR3d
Leu4
Leu3a
2H4
UCHLI
OKT8
OKMI
LeuM5Leu 11Leu 19
RR i/l
lL-2 R
IKIal
T-cell receptor ot/pT-cell receptor y/5
pan T-cell
T-helper/inducerT-subset, B, G, M.
T-subset, B, G, M.T-suppressor/cytotoxicM , G, NK.
Jignifieant difference between the control group and vitiligo patients (P < 0.05).
323
Internationa! Journal of DermatologyVol. 31, No. 5, May 1992
CD4 CD 8
2 30•5
Control Vifiligo
pofients
Control Vitiligo
potients
Figure 3. Percent values of CD4-I- and CD8-1- cells in vitiligopatients and control individuals (P> 0.05).
CD45 RA CD45 RO
60 -
5 50o
30 -<
Control Vitiligopotientj
Control Vifihgopotients
Figure 4. Percent values of CD45RA+ and CD45RO+ cells inthe vitiligo and control group. There is a significant decreasein CD45RA+ cells (P > 0.03) but no significant change inCD45RO+ cells.
with IDD. These changes were attributed to the autoim-mune pathogenesis of IDD, as they were absent in non-insulin-dependent diabetes mellitus (NIDD). In SLE, it hasbeen suggested that anti-T-cell antibodies reactive withCD4+CD45RA+ may play some role in eliminating themfrom the circulation.^' Vitiligo may be associated withdiabetes mellitus., particularly of the IDD type.,* rheuma-toid arthritis/" and SLE.* The presence of similar abnor-malities of circulating T cells in vitiligo patients wouldexplain such an association., if the decrease of CD45RA+affected CD4+ cells. Further investigations are currentlybeing performed to determine whether the decrease ofCD45RA+ cells in vitiligo involves CD4, CDS, or both.
Most of the resting T cells do not express MHC classII or IL-2R antigens. These antigens, however, arerapidly expressed on T cells following activation by anantigen or mitogen.^'-'^ CD54 expression is also associ-ated with T-lymphocyte activation." In our study, nosignificant increment of CD25+ and CD54+ cell popula-tions was found, despite a significant increase of HLA-DR+ expression. An isolated increase of HLA-DR expres-sion without an increase of CD25-expressing cells hasrecently been reported in herpes zoster,^'' pityriasisrosea,^'' and alopecia areata.^^ This discrepancy wasexplained by the in vitro difference in sequential ex-pression of these antigens during T-cell activation.CD25 antigen was expressed within a few hours afteractivation, and its expression was transient. On theother hand, HLA-DR expression was expressed withindays and remained at a high level for a few weeks.•"> By
suits reported in the literature: (1) different stages ofthe disease and different ethnic groups of patients; (2)varying times for obtaining and processing the bloodsamples, a factor that has been carefully avoided inthis study; (3) phenotypically and functionally distinctPBTL subpopulations, e.g., the two functionally differ-ent CD4+ cell subpopulations, CD4+CD45RA+ andCD4+CD45RO+;" (4) cross-reactions of the MoAbs usedwith other types of cells, e.g., Leu-7 reacts not onlywith NK cells but also with a proportion of T and Bcells; (5) variation of techniques (e.g., immunofluores-cence, FACScan), which are not equally sensitive.^'' Inaddition, the borders between the cell subpopulationscharacterized by MoAbs are no longer as rigid as origi-nally thought.^"
There may be some significance in the decrease ofCD45RA+ cells in nonsegmental vitiligo disclosed by thisstudy. A decrease of CD4+CD45RA+ and an increase ofHLA-DR+ cells has recently been reported in autoim-mune diseases, such as insulin-dependent diabetes mel-litus (IDD),^^ rheumatoid arthritis,^*" and SLE.̂ ^ TheCD4+CD45RA+ subset induces CD8+ cells to generate ef-fector suppressor cells, while the CD4+CD45RO+ subsetstimulates B cells for antibody production.^** In addi-tion, an increase of CD25+ cells was found in patients
CD25 CD54
20 -
Control Vitiligo
potienrs
2 0 - 1
3 10 -
o
Control Vitiligo
patients
HLA-DR
0 -I
Control Vitiligo
potients
Figure 5. Percent values of HLA-DR, CD25+ and CD54+ cells invitiligo patients and controls. There is only a significant in-crease in HLA-DR+cells (P > 0.02).
324
Nonsegmcntiil Vitiligo
Abdel-N.iser et al.
CD15 CD55
1 1Control Vitihgo
pafienl5
11
"o
30 -
20 -
10 -
0 -
,,0
— i —
1Control
— ^
1Vihligo
patients
Figure 6. Percent values of the natural killer cell markersCD16+ and CD56+ cells in vitiligo patients and control individ-uals (P>0.05).
CD11 b CD11 c
1 . 0 -
Control Vitiligopofients
Control Vitiligopotients
Figure 7. Percent values of CDl lb+ and CDl lc+ cells in vitili-go patients and control individuals (P > 0.05).
analogy, the normal CD25 levels could be explained bythe fact that most of the patients included in this studyhave had the disease for more than 1 year.
There is no evidence indicating the presence of cir-culating anti-T-lymphocyte antibodies or immunecomplexes in patients with nonsegmental vitiligo. It isunlikely that the decrease of CD45RA+ is due to an in-flux of these cells in the tissues.'̂ -'** Moreover, it hasbeen shown that CD45RO+ cells are the predominantsubset in inflammatory lesions.'^ Consequently, it maybe possible to postulate that the decrease of CD45RA+ isrelated more to the cause than to the outcome of thedisease. Further investigations of vitiligo patients withspontaneous repigmentation and others in longstand-ing remission are necessary for further elucidation.
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Where Did Syphilis Originate.'
Any inquiry into the origin of syphilis encounters two questions: (i) Was there nosyphilis in Europe and the rest of the Old World until tbe end of the fifteenth cen-tury? And (ii), did the men in Columbus' ships bring it from the New World in1493? While Morison (1), biographer of Columbus, says that many medical au-thorities and probably most laymen believe in tbis "American origin" of syphilis,other experts, unsure of the "Columbus theory," regard the origin of syphilis as acomplicated and controversial enigma.
In recent decades, however, much has been learned about spirochetal infection(the disease became treponematosis when the spirochete was renamed the tre-poneme). This accumulating information about the natural history of treponemalinfection and its various manifestations in man has opened up a broader perspec-tive on the question of its origin. Hence, it now seems clear that treponematosiswas in existence on every continent before recorded bistory, pre-dating Columbusby thousands of years. Whatever infection Columbus' men did or did not have,would thus have no bearing on the history of syphilis. From Hudson EH.Christopher Columbus and the history of syphilis. Acta Trop 1968; 25:1-16.
