L~fe Sciences, Vol. Printed in the USA
51, pp.
2107-2116
Pergamon
Press
THALIDOMIDE AND THE IMMUNE SYSTEM 2. CHANGES IN RECEPTORS ON BLOOD CELLS OF A HEALTHY VOLUNTEER
Remhard Neubert*, Ana Cnstma Nogue~ra, Dlether Neubert Institute of Toxicology and Embryopharmacology, and *Pediatric Hospital (Kalserm-Auguste-V,ctorla Haus), Umverslty Chnlc Rudolf Vlrchow, Free Unwerslty Berlin Garystrasse 5, W-1000 Berhn-33 (Received
in fanal
form October
23,
1992)
Summary Thalidomide (Thd) was given in two trials (total dmly dose. 5 or 8 mg Thd/kg body weight, respecuvely) for five and three days to a healthy male volunteer, and various receptors were analyzed on white blood cells before, dunng and after (up to 30 days) the treatment period. There were neither marked dewatlons m the absolute number of total leukocytes nor m the percentage of total lymphocytes or monocytes throughout the study period The most pronounced changes were observed in the surface receptors on CD4 ("helper cells") cells and leukocytes bearing the C D l l b (Mac 1) and other lntegrln and adhesmn receptors Other changes included shifts m the ratio cytotox~c cells/suppressor cells as well as a reduction of the receptor density (passage from bright to dim) m T helper cells beanng CD45RO "memory" markers. S~multaneously, the number of B cells was found to be increased as was the percentage of some adhesion receptors on CD8 + cells Unhke in prewous experiments in which Thd was administered to marmoset monkeys, no effect could be seen in cells bearing the CD2 (LFA-2) epltope In previous pubhcatlons [1-5] we have described defined changes reduced on lymphocyte subtypes in the blood of a non-human primate (Calhthrocjacchus) by thahdomlde (Thd) (2-[2,6dmxoplperldme-3yl]-phthahmxde) or EM12 (N-phthallmldmoglutarlmlde), a teratologlcally even more potent Thd derivative which has the advantage of breahng down into fewer hydrolysis products or metabohtes A characteristic alteration observed m the blood of marmoset monkeys after treatment with Thd or EM12 was a highly reduced reaction of T cells with the monoclonal antibody for the CD2 (LFA-2) receptor. Furthermore, the e p l t o p e density of other f~2 mtegrlns, such as LFA-1 ( C D l l a +, CD18+), decreased or these receptors were no longer detectable on some cells of the treated ammals. This effect was studied in this non-human primate after eight days of treatment, and some of the alterations persisted for more than three weeks after d~scontmuat~on of the treatment We have now investigated whether slmdar alterations to those seen m the marmoset monkey can also be reduced by Thd m man. Here we report on the changes induced by Thd in the white blood cells of a healthy volunteer. These data were collected to serve as a basxs for selecting the appropriate variables for further, more extensive studies in man on effects of Thd under normal and pathological conditions.
Data *n this paper are part of the doctoral thesis of Aria Cnstma Nogue*ra, to be submitted either to FIOCRUZ, Rio de Janeiro, Brazal or to the FU Berlm, FRG
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0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rlghts reserved.
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1992
Materials and Methods It was attempted to select experimental conditions similar to those of the thelapeut~c use of Thd and comparable to the previous studies performed in marmoset monkeys Volunteer and dosing schedule: In the first trial venous blood was obtmned shortly before 09.00 hours on nine days from a healthy 62 year old male volunteer (body weight 88 kg, height" 180 cm) directly before, during and after the 5-day dosing period Pure Thd (micromzed) was taken orally as a powder (5 mg/kg body wt dmly), divided into three doses taken at 8 hour intervals (09:00, 17:00, 01:00 hrs). Aside from a dry mouth, constipation, and a feeling of sleepiness and dizziness 3 to 5 hrs after the dosage, no adverse effects were noted, there was especially no "hang-over" in the morning from the dose taken after midnight Three months after the end of the first treatment period, a second tnal was lnalated with the same volunteer, using somewhat higher doses (8 mg Thd/kg body wt as total dally dose) to verify and intensify the effects observed before In this trial, Thd was taken in five doses per day (08 00, 12 00, 16:00, 20:00, 24.00 hrs). During the first day of treatment (lmtlal dose addmonally at 03.00 hrs) white blood cells were analyzed for possible effects already after 6 hrs (1 dose) and 12 hrs (3 doses). The last (17 th) dose was gwen at 04"00 hrs on the 4 th day, and the last blood sample taken 4 hrs later (77 hrs after the begin of the treatment period) Blood cell analysis: The blood was ammedmtely transferred from the syringe into 4 ml tubes coated with EDTA dl-potasslum (Gremer, Nurntmgen, FRG) and gently mixed Concentrations of leukocytes and lymphocytes were measured with a Sysmex F800 Mlcrocellcounter (TOA Medical Electronics, Hamburg, FRG), and additionally, the percentage of various white blood cells was calculated from differential blood cell counts
Analyses were performed with the whole blood. Samples of 50 #1 blood were reacted wnh optimal concentrations of (a mixture of) the specific monoclonal anUbodles for 15 mln at 4°C in the dark, the erythrocytes were lysed (Dlanova lysls reagent, lot # 10, 1 ml - 1 25), and the cells fixed by adding 250 /A Dlanova fixing reagent (lot # 10) After washing the cells, lymphocyte subpopulatlons were measured using a FACScan (Becton-Dlcklnson, Heidelberg, FRG) Gating was performed on the lymphocyte, monocyte and granulocyte populations respectively, determined by thetr dlStlnctwe forward and side scatter charactenstlcs The results are expressed as percentage of the so-defined populations. Surface markers were analyzed with W m D s t 1 0 software obtained from Verity Software House (Topsham, Me ) The following monoclonal antibodies (mAbs) against human epltopes were used in the study" CD2 (Tll, FITC lot # 278El14, PE lot # 940F174), CD5 (T1, PE lot # 1341KI14), CD7 (3AI, FITC lot # 2309C014), CD11b (Mol, FITC lot # 0129F144), CD14 (Mo2, FITC lot # 458M114, PE lot #1658C034), CD19 (B4, FITC lot # 1618F054, PE lot # 1069K084), CD20 (B1, BI lot # 0448L094), CDw21 (B2, lot # PE lot # 1219F084), CDw29 (4B4, FITC lot # 1531E064, PE lot # 1221A154), CD33 (My9, FITC lot # 811G043), CD45RA (2H4, FITC lot # 1180F054, PE lot # 1171A154), CD56 (NKH1, PE lot # 0900M154), from Coulter Electronics (Krefeld, FRG), CD3 (leu4, PerCP lot # R0416), CD4 (leu3a, PerCP lot # R0301), CD8 (leu2a, PerCP lot # R0256), CDIO (Calla, FITC lot # P0150), CDlla (LFA-I~, FITC lot # R0272), CD11c (leuM5, PE lot # P1267), CD16 (leul la, FITC lot # N0666), CD18 (LFA-I/~, FITC lot # P0516), CD20 (leul6, PerCP lot # R0262), CD23 (leu20, PE lot # P0405), CD25 (IL2-R, PE lot # P0618), CD28 (leu28, PE lot # P0768), CD38 (leul7, PE lot # R0419), CD44 (leu44, FITC lot # P0432), CD45RA (leul8, FITC lot # P1162), CD45RO (leu45RO, PE lot # R0467), CD54 (leu54, PE lot # R0543), CD57 (leu7, FITC lot # N0911), CD69 (ieu23, FITC lot # P0518), HI_M-DR (HLADR, FITC lot # N0720, PE lot # P0941), HLA-DQ (HLA-DQ, FITC lot # N0812) from Becton Dickinson (Heidelberg, FRG) CD38 (PlasmaC, IOB6, lot # 07), CDw49b (VLA tx2, IOP49b lot # 04), CDw49d (VLA oe4, 10P49d lot # 05), CD58 (LFA-3, IOT58, lot # 04) from Dlanova (Hamburg, FRG)
Vol.
51, NO. 26, 1992
Thal~domlde
and the Immune System
2109
The monoclonal antibodies (mAbs) used were fluorescemlsothiocyanate (FITC)-, phycoerythrlne (PE)-, or pendinm chlorophyll protein reagent (PerCP T) -labelled and incubated with the lymphocytes for 15 mm at 4°C m the presence of 0.1% sodium azlde. Sixty-eight different combinations of mAbs m triple-labelled assays were analyzed with the FACScan for each of the 14 blood samples m this study. Several of the values were cross-checked from the various mAbcombmatmns. Chemicals: Thahdomlde (2-[2,6-dloxoplperidine-3yl]-phthallmlde), was kindly provided by Dr. Ernst Frankus (Grunenthal GmbH, Stolberg, FRG) as a white micromzed powder (lot # 1057/A), and had a punty of 99%, according to the manufacturer.
Overview on Some of the Surface Markers Analyzed on the Various Cell Subpopulat~ons Characterized by surface markers
Cell subpopulatlon pan T T, activated T, actwated (IL-2 receptor) T, E-posmve pan helper (CD3+CD4 +) Helper-reducer ("memory") Helper-inducer, actwated Suppressor-inducer ("naive") pan suppressor/cytotOXlC Suppressor Cytotoxlc T, N-CAM bearing subset Cytotoxlc T, subset T cells with "lymphocyte homing receptor"
CD3 + CD3 +HLADR + CD3 + CD25 + CD2 + CD4 + CD4 + CD45R0 + CD45RACD4 + CD45R0 + CD45RA-CDw29~on) + CD4 + CD45RA(br0 + CD45R0 CD8 + CD8+CD56 CD8+CD56 + CD8 +CD57 + CD3+leu8 +
Natural kdler,cytotoxlc T cell subset Natural kdler, subset mature B
CD3"CD56 + CD3-CD16 + CD19 +
Monocytes
CDI4 +
Cells with some selected adhesion receptor~ Helper wsth LFA-h~ Helper with ICAMI Helper with [~PA}Ifl Helper with
CD4+CDlla + CD4+CD54 + CD4+CD44 + CD4+CD18 +
Helper-inducer (",memory',') with LFA-la Helper-reducer (' memory ') with LFA-1B Helper-inducer ("memory") with Pgp-1
CD4+CD45R0+CD1 la + CD4 + CD45R0 + CD 18 + CD4 + CD45R0 + CD44 +
Suppressor/Cytotoxlc T with LFA-la Suppressor/Cytotoxlc T with ICAM-1
CDS+CDlla + CD8+CD54 +
Monocytes with LFA-I~ Monocytes with Mac 1 Monocytes with p 150,95
CD14+CDlla + CD14+CDllb + CD14+CDllc +
Results In the tables the deviations from the 0-time values by a factor > 2 are indicated as framed numbers. These values are certmnly outside any normal variation. Numerous alterations in the surface receptors were observed dunng the course of the treatment, some effects became ev,dent (see. Table II) after only 6 hrs following the first dose (1.6 mg Thd/kg body wt).
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1992
Total white blood count: As may be seen from the data compiled m Table 1, there were only minor deviations m the number of total leukocytes or total lymphocytes and monocytes per/xl blood during the course of the study. Effects on lymphocyte receptors: One of the first effects observed (see Tables I and II) concerns the epltope density of some ga lntegrln receptors (e g LFA-lc~, LFA-lg) on the surface of certain cell types (e.g CD4+CD45R0 + cells), as indicated by the change bright --, &m Th~s Is followed by a total loss of these receptors on many cells and the occurrence of mtegnn minus cells. Such an effect is dlustrated m the onglnal FACScan dmgram shown m Fagure 1 for the C D l l a (LFA-lc0 receptor It is obvious that during the treatment period more and more cells appear which do not bear the high epRope density L F A - l a receptor (positive cells = red, negative cells = black). Closer analyses reveals that this effect ~s more pronounced on CD4 "helper" cells than on the CD8 "suppressor/cytotoxlc" subset. Note that the d~sappearance and subsequent reappearance of L F A - l a on the monocytes ~s accelerated after the treatment period when compared to the lymphocyte fraction. A second lntegrln, CDllb(bn), shows a samflar effect on the lymphocyte fraction as LFA-lc~ The third member of the antegnn family to be found on lymphocytes, LFA-2 (CD2, E + rosettmg receptor) does not react in a s~mflar way Where the &sappearance of the CD2 receptor and subsequently the appearance of cells CD4+CD2 - constituted one of the most spectacular effects m the marmoset monkey after treatment with Thd and its denvatwes, no noticeable effect could be seen m the human parallel This appears to be one of the few major differences an the human vs marmoset experaments Other adheslon molecules (CD44, CD54) show s~mdar "down-regulatory" effects m thear receptor expressmn though here, the effect seems to be more evenly &stnbuted over the entire leukocyte fractmns and thus more difficult to pro-point to one specific cellular subtype In course of the Thd treatment yet another difference could be found m the CD4 subset Where before treatment < 2% cells existed beanng CD4+CD45RO-CD45RA-, dunng and after the treatment period these cells could be found much more frequently (see table II) Ad&tlonally, the receptor expression of CD45RO showed signs of "down-regulataon", many of the posmve cells showed this "memory" receptor in low to very low receptor density Of the two "lymphocyte homing receptors" investigated in th~s study (CD44 and Leu8) only CD44 showed a reaction to the treatment Leu8 bearing cells showed no change No change could equally be noted in the cytotox~c subset CD3+CD57 + Only a rather shght decrease could be noticed an the C D l l c (p150.95) g2 lntegnn. WRhm the lntegrln family th~s was by far the least spectacular effect A significant increase of B cells (CD19) could be seen in both trials These findings were more pronounced m the second tnal. Actwatlon markers (HLA-DR and CD25 the mterleukm 2 receptor) decreased sensibly during the treatment
Effects on monocyte receptors: Pronounced changes were also observed on the surface markers of monocytes. As early as 6 hours after the beginning of treatment cells appeared m the monocyte fractmn which were not beanng Mac l(brl) epltopes as charactenzed by CD1 lb(bri) In the course of the treatment, this receptor mtenslty almost completely &sappeared from the cell surface of these cells, leaving cells with no or very low receptor density After the end of the treatment, however, the density expression increased astonishingly fast m comparison with other mtegnns on the lymphocyte fractmn for instance W~thln 29 hours after the last dose, the monocytes again showed more than 40% cells beanng thas receptor. This appears to be the most drastic and the most rapidly reversing effect seen on any of the three cell fractions studied Effects on granulocyte receptors: A clear-cut decrease m the number of cells carrymg defined mtegnn receptors could also be demonstrated in the granulocyte fractmn This ~s illustrated in Figure 2 for C D l l b .
Vol. 51, No. 26, 1992
CD1
Thalidomide and the Immune System
2111
Fwre 1 : Orrglnal dotplot,CDl 1a bright posttlve cells were color backgated and plotted by their foreward and side scatter parameters
l&r
(LFAla)
control sampling before treatment wrth thalld
granulocytes monocytes lymphocytes
at 6 hrs (1 dose) 4-l
12 hrs after last dose -
2112
Thalldomlde
and the Immune
Vol.
51, No.
26,
1992
Ft~ure 2
C D 11 b br,ght (Mac-l,
System
Original dotplot, CD1 l b br'~"t posltwe cells w e r e color backgated and plotted by their f o r e w a r d and side scatter parameters
,I
CR3)
control sampling before treatment w~th thahdom~de
granulocytes monocytes lymphocytes 100
20o
300
400
5 r)
600
700
800
900 100(
~Z at 6 hrs (1 dose)
,
¢ o~ I
at 12 hrs (3 doses)
o~ r,
r 100
lerTIfH~ 200 300
I[ 400
500
7 Irr rl~ I [I r[ 600 ZOO 8OO #00 100r
1 oo
2nLt
t
}:
at 29 hrs (6 doses)
:r
I 'I
I at 77 hrs (17 doses)
.4 r T
100
.DO
] ~ W T
JrJ0
4(]0
500
600
7r'0
[
~ r
b0("
1
T--
9(3L. 10Oi
I 12 hrs after last dose
-,,t,
4"
] 29 hrs after last dose
.pl 100
200
300
400
500
600
700
800
~00 100,
~. .-' . . .,'.oo 20or~T~4~o300 5~o ~oo 7oo~aoc. . . .~oo o
Day
70
988 89 7
83 0 612 384 224 17 375 261 173 163 206 22 6 179 151 73 73 57
61 262 1600 244
Before 0*
68
[~ 63 1
87 1 574 380 193 20 379 195 138 112 11 5 12 3 145 1 1 2 80 69 53
54 29 6 1600 324
116
~ 56 4
74 8 557 304 180 17 286 165 164 [~] 10 7 ~ 130 8 3 9 81 76 81
62 22 6 1400 372
Dunng 1'* 3
[]~
~33[~
58 4 518 313 191 16 223 158 118 ~ T3~ ~ 126 5 97 58 75
49 265 1300 345
5
118
~ 64 7
80 7 553 343 193 18 262 207 165 ~ ~ 12 2 173 8 9 102 62 69
46 239 1100 460
After 1"** 63 23 8 1500 504
9
84
874 79 3
87
991 83 6
86 6 86 0 659 635 421 434 192 201 22 22 21 6 267 322 342 172 150 11.4 195 26 7 25 1 25 3 293 152 142 8 0 8 9 5 112 118 6.6 64 67 63
52 231 1200 416
3 52 269 1400 416
31
94
966 83 4
103
990 86 1
89 0 82 5 582 587 399 396 182 164 22 24 233 286 323 248 199 145 171 187 24 0 182 27 7 18 4 155 130 1 6 1 116 103 69 91 63 46
6 1 23 0 1400 549
16
72
974 79 8
86 4 695 520 187 28 21 1 345 233 160 15 6 25 5 126 9674 90 53 44
51 255 1300 408
126
761 56 6
103 39 42
77 2 626 454 177 26 23 1 252 185 139 [6~ 14 2 68
56 28 6 1600 560
Second 0"*** 025
[~q]
~
~
~
6.3
14 1 [55] 15~.
162 [~] [~ 136 47 67
~
79 0 664 510 143 36 173 248 25.6
54 304 1700 448
32
72 2 675 522 168 31 13.6 292 175 159
56 28 6 1600 560
05
Framed: values altered by a factor of>2 when compared with 0-time values Absolute numbers are given as cells/lal blood, percentages of subpopulatlons are from total lymphocytes co in" thousand cells/lal blood Bloodsamphng * shortly before dosing, ** during 5-day-doslngpenod, *** after the endofthe dosing period Blood samphng **** before and during a second dosing period (5 doses dally of 1 6 mgThd/kg body wt)
54 22 2 1200 270
After 12
[~
~
10.2 99 51 70
79 7 626 431 13,1 33 183 228 23.1
Thd was taken 3 times daffy (total dally dose 5 mg/kg body wt) for 5 days Receptors on white blood cells were momtored before, dunng and up to 4 weeks after the treatment period Twelve weeks after the first treatment period the results were confirmed in a second study (wath daffy 8 mg Thd/kg bw), analyses at 6, 12, 29 and 77 hrs after the begm of treatment
%CD19 ÷
B Ivmohocvtes
%CD14*CDllb(bri) . % CD14+CD11a(bri)÷
Monocvtes
% CD2÷ %CD3 ÷ °/o CD4~ %CD8 ÷ Ratio CD4+/CD8+ %CD3 CD56÷ % CD4+CD45RO÷ % CD4+CD45RA+ % CD4"CDw29(briyCD11a(bri) ÷ % CD4*CDw29(bri)÷CDI8(bri) ÷ % CD4*CDw29(bri)+CD44(br0% CD4+CD54* % CD8+CD56÷ % CD8+CD56 %CD8"CD11a(brl) ÷ % CD8+CD54(bn)÷
T Ivmohocvtes
% lymphocytes Totallymphocytes Total monocytes
Total leukocytesor)
Subset
TABLE I Effect ofThahdomlde (Thd) on Surface Receptors o f White Blood Cells in a Healthy Volunteer
bO t.a I-a tad
rt
¢D
¢D
O"
tx O
i-a
0.-]
i..a to tO I,O
O't
< o
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Thal~domxde
and the I m m u n e
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Vol.
51, No.
26,
1992
TABLE II Effect of Thahdomlde on Some Surface Receptors on White Blood Cells Days of treatment Before Durmg 0"*** 0 25 05
Cell subpopulauon T Iymphocytes % CD4+CD45RO+CDlla(dlm) + % CD4+CD45RO+CDlla(bri) + % CD4+CD45RO+CD18 (bri) + % CD4+CD45RO+CD18 (dim) + % CD4+CD45RO+Ieu18 + % CD4+CD45RO(bn)+Ieu18 % CD4+CD45RO(dtm)+leu18 % CD4+CD45RO-leu18 -
22 16 20 13
5 7 4 8
20 10 13 15
8 6 0 8
5 5 19 4 6 6
3 5 15 8 8 9