Abstract

Thalidomide is effective in the treatment of some tumor necrosis factor—related diseases, but its cellular target is not known. Effects of thalidomide were investigated on lymphocytes and monocytes. Cell migration was examined in a Boyden chamber. Effects on protein kinase C (PKC) were investigated functionally by use of PKC inhibitor and in purified enzyme preparations. Thalidomide itself showed no direct chemotactic effect on lymphocytes or monocytes. Preincubation with the drug significantly enhanced random migration of both cell types. This effect was bisindolylmaleimide-reversible, suggesting involvement of PKC. Preincubation with thalidomide diminished the chemotactic response of monocytes towards formyl peptide but failed to influence lymphocyte chemotaxis towards RANTES or interleukin-8. In a cell-free assay, inhibition of PKC activation by bisindolylmaleimide could be reversed by thalidomide, indicating direct interactions of thalidomide with PKC. Results suggest that effects of thalidomide in chronic inflammation may be related to actions on leukocyte functions.

Thalidomide [2-(2,6-dioxo-piperidine-3-yl)-iso-indole-1,3-dione)], a racemic mixture with a turbulent pharmacologic history, has been used as a potential sedative and antiinflammatory drug. It has been effective in treatment of leprosy and other inflammatory dermatoses, such as aphthosis, suggesting a useful role in human immune complex diseases [1–3]. Its immunomodulatory abilities result in beneficial effects on erythema nodosum leprosum, chronic graft-versus-host disease, discoid lupus erythematosus, Behçet's disease, and Langerhans cell histiocytosis [1, 4]. In searching for the cellular targets of thalidomide, several models of immune regulation have been investigated. Direct effects on cell lineages of the immune system include cytokine synthesis [1]; inhibition of mitogen-stimulated lymphocyte proliferation [5]; modulation of adhesion molecules on endothelial cells [1]; and tumor necrosis factor (TNF)—, interleukin (IL)-1—, and lipopolysaccharide-induced neutrophil chemotaxis and transendothelial migration [6]. Others have shown reduced viral replication in a human immunodeficiency virus—infected monocytic cell line treated with thalidomide. The advantage of thalidomide compared with other antiinflammatory drugs is its selective inhibition of TNF production and its wide therapeutic range, although the molecular target has not been identified [1].

Protein kinase C (PKC) is a generic expression for a family of serine/threonine kinases that can be further subdivided into related groups [7]. The activation of the conventional PKCs (α, βI, βII, and γ) is induced by Ca2+ and diacylglycerol or phorbol esters. PKC activation plays an important role in numerous cellular functions, including migration, cell transformation, and vesicular trafficking [8]. Involvement of PKC in cellular signaling pathways can be detected by selective and nonselective PKC inhibitors. 2[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GFX), a selective PKC inhibitor of the substituted bisindolylmaleimide family, reduces leukocyte migration and neutrophil respiratory burst [9].

Because chronic inflammation is characterized by lymphocyte and monocyte tissue infiltration, we attempted to learn whether thalidomide exerts immunomodulation in a manner that may explain the beneficial effects of thalidomide therapy. We investigated in vitro migration of lymphocytes and monocytes treated with thalidomide and its effect on purified PKC.

Materials and Methods

Lymphocyte and monocyte preparation

Peripheral blood mononuclear cells (PBMC) were obtained from forearm venous blood of healthy volunteers (anticoagulated with EDTA) or from buffy coats mixed with phenol red-free Hanks' balanced salt solution without Ca2+ and Mg2+ (Gibco BRL Life Technologies, Vienna) in a ratio of 1:1. After Lymphoprep (Nycomed Pharma, Oslo) density gradient centrifugation, PBMC were collected and washed three times with normal saline. Positive selection of lymphocytes and monocytes was done by adding magnetic cell sorter (MACS) colloidal superparamagnetic microbeads conjugated with monoclonal anti-human CD3 or anti-human CD14 antibodies (Miltenyi Biotec, Bergish Gladbach, Germany) to cooled, freshly prepared PBMC preparations in MACS buffer (PBS with 5 mM EDTA and 0.5% bovine serum albumin [BSA]) according to the manufacturer's instructions. Cells and microbeads were incubated for 15 min at 4°C–6°C. In the meantime, the separation column was positioned in the MACS magnetic field and washed with MACS buffer at room temperature. The cells were washed once with MACS buffer, resuspended, and loaded onto the top of the separation column. The eluent containing CD3 or CD14 cells was withdrawn, and, after removal of the column from the magnet, trapped lymphocytes or monocytes were eluted with 6-fold dilution of cold MACS buffer, centrifuged, and resuspended in RPMI 1640 (Biological Industries, Kibbutz Beit Haemek, Israel) containing 0.5% BSA to a final concentration of 106cells/mL. The preparation was ∼98% pure [10].

Cell migration experiments

Chemotaxis (directed migration) and chemokinesis (random migration with enhanced speed and frequency of locomotion) were measured in a 48-well Boyden microchemotaxis chamber (Neuroprobe, Bethesda, MD) in which a 5-μm pore-sized cellulose nitrate filter (Sartorius, Göttingen, Germany) separates the upper and lower chamber. Lymphocytes and monocytes were treated for 30 min with thalidomide (Grünenthal, Aachen, Germany; 0.01–10 μg/mL), GFX (0.5–1000 nM), a combination of both, or vehicle, then washed twice and resuspended in RPMI 1640-0.5% BSA to a final concentration of 106 cells/mL. We added 50 μL of the cell suspensions to each well, and cells were allowed to migrate toward medium or test substances. Migration time was 90 min for monocytes and 120 min for lymphocytes at 37°C in humidified atmosphere (5% CO2). Thereafter, the nitrocellulose filter was dehydrated, fixed, and stained with hematoxylineosin. Migration depth of cells into the filter was quantified by microscopy, measuring the distance (μm) from the surface of the filter to the leading front of cells. Data are expressed as chemotaxis index (CI), which is the quotient of the distances of stimulated migration divided by random migration of cells into the nitrocellulose filter [10].

Measurement of PKC activity

PKC activity was determined by commercial colorimetric PKC assay kit (Pierce, Rockford, IL). In brief, purified PKC standard preparations (mixture of PKC-α, -β, and γ from rat brain) were incubated with reaction buffer, GFX, or GFX concomitantly with effective concentrations of thalidomide. Dye-labeled PKC pseudosubstrate was added, and after activation as recommended in the assay protocol, samples were centrifuged twice over supplied affinity membranes. The remaining phosphorylated products were washed from the membranes with elution buffer, and absorbance was quantified at 570 nm in an ELISA reader (Labsystems, Helsinki).

Dissolving of thalidomide

As recommended by the manufacturer, thalidomide was dissolved in dimethyl sulfoxide (DMSO) to a stock solution of 40 mg/mL just before use in each experiment. Dilution steps led to a final concentration of 0.25% DMSO for all experiments. For vehicle controls, 0.25% DMSO in appropriate buffers was used.

Statistical methods

Data are expressed as mean and SE. Means were compared by Mann-Whitney U test and Kruskal Wallis nonparametric analysis of variance. P< .05 was considered significant. Statistical analyses were performed using the StatView software package (Abacus Concepts, Berkeley, CA).

Results

Chemotaxis towards thalidomide

To determine whether thalidomide exerts direct chemotactic effects on lymphocytes or monocytes, untreated cells were allowed to migrate toward various concentrations of thalidomide (0.01–10 μg/mL). Regulated on activation, normal T cells expressed and secreted (RANTES; 6.25 nM) and FMLP (10 nM) served as positive controls for lymphocytes and monocytes, respectively. None of the concentrations of thalidomide tested could attract cells, indicating no direct chemotactic effect on lymphocytes and monocytes (data not shown).

Effects of thalidomide on lymphocyte and monocyte migration

Cells were incubated for 30 min in the presence of thalidomide (0.01–10 μg/mL), washed twice with PBS, and tested for migration toward classical chemotaxins. Directed migration of lymphocytes towards RANTES (6.25 nM) or IL-8 (1 nM) remained unaffected by thalidomide preincubation (figure 1). In contrast, thalidomide at concentrations of 0.1–10 μg/mL decreased monocytes migration toward FMLP [10 nM] by ∼50% (figure 1). Of interest, random migration of both cell types was significantly enhanced by thalidomide pretreatment. This effect was more pronounced in lymphocytes than in monocytes: it was bell-shaped in monocytes and followed a straight dose-response relationship in lymphocytes, with the highest concentration of thalidomide tested being most potent (figure 1). In searching for the cellular targets responsible for this effect in lymphocytes and monocytes, we tested GFX, a highly selective PKC inhibitor. GFX at concentrations >50 nM (1000 nM was the highest concentration tested) diminished the stimulatory effect of 10 μg/mL thalidomide on lymphocytes by ∼50% (CI for 10 μg/mL thalidomide = 1.41 ± 0.071; CI for 10 μg/mL thalidomide + 500 nM GFX = 1.13 ± 0.059; n = 7; P< .05). In monocytes, GFX at concentrations as above could abolish the effect of thalidomide at 0.1 μg/mL (CI = 1.21 ±0.047 and 1.01 ± 0.037 for 0.1 μg/mL thalidomide alone and with 500 nM GFX, respectively; n = 6; P < .05). GFX itself at all concentrations tested did not influence random cell migration (data not shown). Directed migration of monocytes against formyl-peptide was canceled by 100 nM GFX. This known effect of FMLP was not significantly affected by 0.1 μg/mL thalidomide (data not shown).

Figure 1

Migration of lymphocytes and monocytes into cellulose nitrate. Cells were pretreated with various concentrations of thalidomide or vehicle for 30 min. Migration depth was quantified microscopically. Data are mean ± SE of chemotaxis index (ratio of directed divided by undirected migration): n = 5 for lymphocytes; n = 6 for monocytes. Statistical analysis: Mann-Whitney U test after Kruskal Wallis analysis of variance; * P < .05; ** P < .01; n.s., not significant.

Figure 1

Migration of lymphocytes and monocytes into cellulose nitrate. Cells were pretreated with various concentrations of thalidomide or vehicle for 30 min. Migration depth was quantified microscopically. Data are mean ± SE of chemotaxis index (ratio of directed divided by undirected migration): n = 5 for lymphocytes; n = 6 for monocytes. Statistical analysis: Mann-Whitney U test after Kruskal Wallis analysis of variance; * P < .05; ** P < .01; n.s., not significant.

Interactions of thalidomide with PKC

Because results from the migration experiments indicated involvement of PKC in thalidomide effects, we tested to see whether thalidomide can directly affect PKC activity, using purified enzyme preparations. Thalidomide (0.01 and 1 μg/mL) slightly, but not significantly, produced nearly maximal activation of the enzyme (1.0 U/mL PKC) in the assay system; GFX (100 and 500 nM), as expected, prevented PKC activation. Thalidomide at both concentrations failed to affect inhibition induced by 500 nM GFX but dose-dependently reversed effects of 100 nM GFX on 1.0 U/mL purified PKC (table 1).

Table 1

Effects of thalidomide (THD) and bisindolylmaleimide on a purified enzyme preparation of protein kinase C (PKC).

Table 1

Effects of thalidomide (THD) and bisindolylmaleimide on a purified enzyme preparation of protein kinase C (PKC).

Discussion

The involvement of different leukocytes in inflammatory processes leads to the development of acute or chronic inflammation. Unlike acute inflammatory events in which neutrophil granulocytes play a major role, chronic inflammation is characterized by migration of lymphocytes and monocytes into the affected tissue [11]. The transformation of mononuclear phagocytes into multinucleated giant cells and epithelioid cells results in the formation of granulomas that are specific for several chronic inflammatory diseases [12]. In such situations thalidomide has been said to exert beneficial effects and has been successfully used to treat patients with erythema nodosum, rheumatoid arthritis, and sarcoidosis [1].

Leukocyte migration is a complex phenomenon that includes cell activation for chemokinesis and polarization and orientation in the direction of the highest chemoattractant concentration for chemotaxis. Signal transduction pathways for these processes are not yet completely understood [13]. Mechanisms of action of antiinflammatory drugs on the cellular level include inhibition of cytokine production or direct effects on cell migration. Although the therapeutic efficacy of thalidomide treatment in patients with erythema nodosum leprosum is considered to be due to an inhibition of TNF-α, the mechanism in other thalidomide-sensitive diseases is comparatively unknown [14].

In our experiments, preincubation with thalidomide inhibited monocyte, but not lymphocyte, chemotaxis. In the absence of a chemotactic gradient, the drug itself enhanced undirected cell migration, suggesting chemokinetic effects of thalidomide on lymphocytes and monocytes. This effect was stronger and directly dose-dependent in lymphocytes. Therefore, we continued searching for the cellular targets in leukocytes. Since GFX is a highly selective and potent inhibitor of several PKC isoforms and functionally abolished thalidomide effects in our migration experiments, we rendered PKC a primary target of thalidomide. For confirmation of this hypothesis, we further investigated interactions of thalidomide with purified PKC preparations. Because of nearly maximal stimulation of the enzyme in the assay kit, thalidomide showed little additional activation. However, thalidomide significantly reversed GFX-induced inhibition of PKC.

Taken together, it is most likely that thalidomide directly interacts with PKC in a manner that could stimulate enzyme activity. This would in part explain the chemokinetic properties of thalidomide in lymphocytes and monocytes and a lack of action on RANTES and IL-8—induced chemotaxis in lymphocytes, since both chemotaxins primarily involve phospholipases, not PKC. In chemotaxis induced by formyl peptides, PKC is directly involved in signal transduction [15], and thalidomide inhibited this response (figure 1). Additional activation or intrinsic action by thalidomide on PKC may result in the net effect of inhibition, as previously shown with phorbol esters [15].

The therapeutic efficacy of thalidomide may be due to its immunomodulatory properties on lymphocytes and monocytes within chronically inflamed tissue, and the involvement of PKC in thalidomide-induced effects on leukocytes may be a step toward better understanding the pharmacodynamics of the drug.

Acknowledgments

We thank Grünenthal Inc. for providing thalidomide.

References

1.
Zwingenberger
K
Wnendt
S
Immunomodulation by thalidomide: systematic review of the literature and of unpublished observations
J Inflamm
 , 
1995
, vol. 
46
 (pg. 
177
-
211
)
2.
Hendler
SS
McCarty
MF
Thalidomide for autoimmune disease
Med Hypotheses
 , 
1983
, vol. 
10
 (pg. 
437
-
43
)
3.
Fauré
M
Thivolet
J
Gaucherand
M
Inhibition of PMN leukocytes chemotaxis by thalidomide
Arch Dermatol Res
 , 
1980
, vol. 
269
 (pg. 
275
-
80
)
4.
Vogelsang
GB
Farmer
ER
Hess
AD
, et al.  . 
Thalidomide for the treatment of chronic graft-versus-host disease
N Engl J Med
 , 
1992
, vol. 
326
 (pg. 
1055
-
8
)
5.
Keenan
RJ
Eiras
G
Burckart
GJ
, et al.  . 
Immunosuppressive properties of thalidomide: inhibition of in vitro lymphocyte proliferation alone and in combination with cyclosporine or FK506
Transplantation
 , 
1991
, vol. 
52
 (pg. 
908
-
10
)
6.
Dunzendorfer
S
Schratzberger
P
Reinisch
N
Kähler
CM
Wiedermann
CJ
Effects of thalidomide on neutrophil respiratory burst, chemotaxis, and transmigration of cytokine- and endotoxin-activated endothelium
Naunyn Schmiedebergs Arch Pharmacol
 , 
1997
, vol. 
356
 (pg. 
529
-
35
)
7.
Dekker
LV
Parker
PJ
Protein kinase C—a question of specificity
Trends Biochem Sci
 , 
1994
, vol. 
19
 (pg. 
73
-
7
)
8.
Bacon
KB
Schall
TJ
Dairaghi
DJ
RANTES activation of phospholipase D in Jurkat T cells: requirement of GTP-binding proteins ARF and RhoA
J Immunol
 , 
1998
, vol. 
160
 (pg. 
1894
-
900
)
9.
Toullec
D
Pianetti
P
Coste
H
, et al.  . 
The bisindoylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C
J Biol Chem
 , 
1991
, vol. 
266
 (pg. 
15771
-
81
)
10.
Dunzendorfer
S
Rothbucher
D
Schratzberger
P
Reinisch
N
Kähler
CM
Wiedermann
CJ
Mevalonat-dependent inhibition of transendothelial migration and chemotaxis of human peripheral blood neutrophils by pravastatin
Circ Res
 , 
1997
, vol. 
81
 (pg. 
963
-
9
)
11.
Imhof
BA
Dunon
D
Basic mechanism of leukocyte migration [review]
Horm Metab Res
 , 
1997
, vol. 
29
 (pg. 
614
-
21
)
12.
Williams
GT
Williams
WJ
Granulomatous inflammation—a review
J Clin Pathol
 , 
1983
, vol. 
36
 (pg. 
723
-
33
)
13.
Lee
J
Ishihara
A
Theriot
JA
Jacobson
K
Principles of locomotion for simple-shaped cells
Nature
 , 
1993
, vol. 
362
 (pg. 
167
-
71
)
14.
Haslett
PA
Corral
LG
Albert
M
Kaplan
G
Thalidomide costimulates primary human T lymphocytes, preferentially inducing proliferation, cytokine production, and cytotoxic responses in the CD8+ subset
J Exp Med
 , 
1998
, vol. 
187
 (pg. 
1885
-
92
)
15.
Kanaho
Y
Nishida
A
Nozawa
Y
Calcium rather than protein kinase C is the major factor to activate phospholipase D in FMLP-stimulated rabbit peritoneal neutrophils. Possible involvement of calmodulin/myosin L chain kinase pathway
J Immunol
 , 
1992
, vol. 
149
 (pg. 
622
-
8
)
Presented in part: International Symposium on Critical Care Medicine; 13th annual meeting, 16–21 November 1998, Trieste, Italy (abstract 30); Wiener Intensivmedizinische Tage; 17th annual meeting, 25–27 February 1999, Vienna (abstract 23).