Thalidomide Increases Human Keratinocyte Migration andProliferation

Maria R. Nasca,1 Edel A. O’Toole,* 1 Prema Palicharla,* Dennis P. West,* and David T. Woodley*Dermatology Clinic, University of Catania, Catania, Italy; *Department of Dermatology, Northwestern University, Chicago, Illinois, U.S.A.

Thalidomide is reported to have therapeutic utility in more than 2-fold in the colloidal gold assay and morethan 3-fold in the scratch assay over control. Althoughthe treatment of pyoderma gangrenosum, Behcet’s

disease, aphthous ulcers, and skin wounds. We investi- pro-migratory, thalidomide did not alter the level ofmetalloproteinase-9 secreted into culture medium.gated the effect of thalidomide on human keratinocyte

proliferation and migration, two early and critical Thalidomide did, however, induce a 2–4-fold increasein keratinocyte-derived interleukin-8, a pro-migratoryevents in the re-epithelialization of skin wounds. Tha-

lidomide at concentrations less than 1 µM did not affect cellular autocrine factor. Human keratinocyte migra-tion and proliferation are essential for re-epithelializ-keratinocyte viability. Using a thymidine incorporation

assay, we found that thalidomide, at therapeutic con- ation of skin wounds. Interleukin-8 increases humankeratinocyte migration and proliferation and is chemo-centrations, induced more than a 2.5-fold increase in

the proliferative potential of the cells. Keratinocyte tactic for keratinocytes. Therefore, thalidomide maymodulate keratinocyte proliferation and motility by amigration was assessed by two independent motility

assays: a colloidal gold assay and an in vitro scratch chemokine-dependent pathway. Key words: cytokine/re-epithelialization/wound healing. J Invest Dermatol 113:720–assay. At optimal concentrations, thalidomide

increased keratinocyte migration on a collagen matrix 724, 1999

T by demonstrating that it inhibited basic fibroblast growth factor-induced corneal neovascularization in rabbits (D’Amato et al, 1994).

The effect of thalidomide on leukocytes has been extensivelyinvestigated in vitro; however, there are no studies in the literatureregarding its effect on keratinocytes (Neubert et al, 1996). Thalidom-ide has been demonstrated to be of therapeutic benefit in a variety

halidomide [(N)-phthalimidoglutarimide] is a syn-thetic derivative of glutamic acid. It was widelyprescribed in the late 1950s as a sedative-hypnotic,but was withdrawn from the market in the early1960s because it was found to be a potent teratogen

(Ochonisky and Revuz, 1994; Tseng et al, 1996). Sheskin (1965,of mucocutaneous disorders including aphthous ulceration, Behcet’s1980) fortuitously found that it was extremely effective in thedisease, and pyoderma gangrenosum (Rustin et al, 1990; Gardner-treatment of erythema nodosum leprosum. Subsequently, severalMedwin et al, 1994b; Jacobson et al, 1997; Hecker and Lebwohl,other dermatoses have also been reported to be responsive to1998). It has been assumed that the beneficial effect of thalidomidethalidomide. Consequently, there has been a growing interest inupon these conditions is due to the drug’s anti-inflammatorythis drug, whose mechanism of action is not yet fully known orproperties as it reduces polymorphonuclear leukocyte chemotaxisunderstood (Grosshans and Illy, 1984; Naafs and Faber, 1985;and phagocytosis, and also inhibits soluble inflammatory mediatorsGardner-Medwin and Powell, 1994a).such as prostaglandins (Nasca et al, 1999). Another commonThalidomide has anti-angiogenic, anti-inflammatory, and immu-denominator to many conditions in which thalidomide has anomodulating properties. It inhibits leukocyte chemotaxis andtherapeutic benefit, however, appears to be ulcerative skin wounds.phagocytosis, lysosome degranulation, and mast cell activationBecause, the healing of cutaneous ulceration requires re-epithelializ-(Barnhill et al, 1984). It also induces a decrease in the T helper/Tation, we wondered if one of the beneficial effects of thalidomidesuppressor ratio of T cells (Gad et al, 1985; Moncada et al, 1985;might be directly upon keratinocyte motility and proliferation, twoShannon et al, 1992; Nogueira et al, 1994). The immunologicearly and critical events in resurfacing skin wounds. A few hourseffects of thalidomide may depend on the inhibition of tumorafter wounding, keratinocytes migrate laterally over the woundnecrosis factor-α (Sampaio et al, 1991; Moreira et al, 1993), andbed. Approximately 1 d later, the cells at the edge of the epitheliumupon the downregulation of several cell surface adhesion markersbegin to proliferate and contribute cells to the advancing tongueon blood leukocytes (Neubert et al, 1992, 1993; Nogueira et al,of migrating keratinocytes involved in resurfacing the wound1994). The anti-angiogenic effect of thalidomide was confirmed(Odland and Ross, 1968; Woodley et al, 1993). We thereforehypothesized that thalidomide might increase keratinocyte migra-tion and/or proliferation, as these are two fundamental steps in re-

Manuscript received July 16, 1998; revised May 20, 1999; accepted for epithelialization of healing wounds.publication July 15, 1999.In this study, we used two independent in vitro migration assaysReprint requests to: Dr. David T. Woodley, Division of Dermatology,

to examine the effects of thalidomide on human keratinocyteUniversity of Southern California, Ambulatory Health Center, 1355 Sanmigration on type I collagen, a matrix molecule found in thePablo Street, Los Angeles, CA 90033, U.S.A.wound bed. We also examined the effect of thalidomide on the1M.R. Nasca and E.A. O’Toole contributed equally to this work

Abbreviation: MMP, metalloproteinase. proliferative potential of human keratinocytes.

0022-202X/99/$14.00 · Copyright © 1999 by The Society for Investigative Dermatology, Inc.

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MATERIALS AND METHODS experimental condition. Each experiment was repeated three times andthe counts were averaged.

Materials Rat-tail collagen I was obtained from Collaborative Biochem-icals. Trypan blue 0.4% was obtained from Sigma (St Louis, MO). Safety Gelatin zymography Keratinocytes were plated on tissue-culture plasticSolve was obtained from Research Products International (Mount Prospect, at a density of 20,000 cells per cm2. After the cells attached, unattachedIL). Tritiated thymidine was obtained from ICN Biomedicals. Thalidomide cells were aspirated and thalidomide was added at various concentrations(a racemic mixture of D[1] and L[–] forms was supplied by Pediatric (0.001–100 nM). Media were harvested after 12 h, centrifuged at 4°CPharmaceuticals Incorporated (Westfield, NJ). Thalidomide was initially (1500 3 g) for 10 min to remove cells and debris, and concentrated 5-foldsolubilized in dimethylsulfoxide and further dilutions were made in with a Centricon 30 (Amicon, Beverly, MA). The effect of thalidomidekeratinocyte serum-free medium. Mitomycin-C was obtained from Sigma. on metalloproteinase-9 (MMP-9) secreted into the media was evaluated

by gelatin-ladened zymography as previously described (Sarret et al, 1992a).Cell culture Human keratinocytes obtained from neonatal foreskins Triplicate experiments were performed.were cultured in low calcium (0.09 mM), serum-free MCDB 153 medium(Gibco BRL, Gaithersburg, MD) as described by Boyce and Ham (1983), Interleukin (IL)-8 protein determination Human keratinocytes wereand modified by O’Keefe and Chiu (1988). All experiments were performed cultured in basal medium prior to trypsinization, then plated on tissueusing human keratinocytes between second and fourth passages. Ker- culture plastic at a concentration of 15,000 cells per cm2. After the cellsatinocytes from 10 donor foreskins were used in this study. At least three attached, increasing concentrations of thalidomide were added to the dishesdifferent donor foreskins were used in each experiment. (0.001–100 nM), and the cells were incubated for 12 h. The concentration

of IL-8 in the conditioned medium was then measured using a commerciallyCell viability available enzyme-linked immunosorbent assay (Quantikine; R&D Systems,Lactate dehydrogenase cytotoxicity assay To quantitate the cellular toxicity of Minneapolis, MN) able to detect 4.7 pg IL-8 per ml. Standard curves forthalidomide, we used a commercially available Cytotoxicity Detection Kit recombinant human IL-8 ranging up to 2000 pg per ml were run for eachfrom Boehringer-Mannheim. This is a colorimetric assay which measures assay. Protein levels in the supernatants were normalized to total cellthe lactate dehydrogenase activity released from damaged cells. Ker- number. Each supernatant was analyzed in triplicate. This experiment wasatinocytes were plated in 24 well plates (5000 cells per well), allowed to repeated three times.attach and then treated with increasing concentrations of thalidomide for16 h. The medium was harvested and the assay was performed as described RESULTS(Racher et al, 1990). Percentage cytotoxicity was calculated from the

Concentrations of thalidomide , 1 µM do not affect ker-mean of triplicate absorbance values using a formula incorporating theatinocyte viability Keratinocyte viability after exposure to tha-experimental value, low control and high control.lidomide was examined by calculating the percentage of nonviable

Cell migration assays cells using an lactate dehydrogenase release cytotoxicity assay. AsColloidal gold assay The method of Albrecht-Buehler (1977), as modified shown in Fig 1, increasing cytotoxicity was observed with increas-by Woodley et al (1988), was used to evaluate keratinocyte motility. ing concentrations of the drug, whereas concentrations , 1 µMCoverslips were coated with colloidal gold salts, washed, and type I collagen minimally affected cell viability. Because keratinocyte cytotoxicity(8 µg per ml) in Hanks’ balanced salt solution supplemented with calcium

was observed at concentrations of thalidomide . 1 µM (Fig 1),and magnesium was added to each well. Dishes were incubated at 37°Cwe used thalidomide at concentrations between 0.001 and 100 nMfor 2 h to permit immobilization of collagen to the coverslips. Passage 3for all biologic assays in this study.keratinocytes were plated in each well at a density of 750 cells per cm2.

After attachment, media containing various concentrations of thalidomideThalidomide increases keratinocyte migrationwere added to each well (0.001–100 nM). Triplicate wells were used forin vitro Human keratinocytes were exposed to a substratum ofeach experimental condition. Cultures were incubated at 37°C, 5% CO2type I collagen (8 µg per ml). The keratinocytes are maximallyfor 16 h, washed and fixed in 0.1% formaldehyde in phosphate-bufferedstimulated to migrate on a type I collagen matrix of 15 µg per mlsaline. Because the doubling time of keratinocytes under these conditions

is approximately 30 h, the assay assesses cellular motility unconfounded by or greater. This optimal matrix provides migration indices abovecell division. To quantitate migration, cells were examined with an Olympusinverted microscope under dark-field optics and the percentage of eachfield occupied by migration tracks, a so-called migration index, wascalculated using computer-assisted image analysis (Chen et al, 1993). Theimages of 10 fields per condition were analyzed.

In vitro scratch assay A second migration assay, the so-called ‘‘scratch assay’’was performed according to Cha et al (1996). Keratinocytes (40,000 percm2) were plated in tissue culture dishes, and after 24 h of incubation, thecells were incubated with 10 µg per ml of mitomycin-C for 2 h to inhibitcell proliferation, and the confluent monolayer of cells was scratched in astandardized manner with a plastic apparatus to create a cell-free zone ineach well, 2 mm in width. The medium was aspirated and replaced withkeratinocyte medium with or without the presence of thalidomide (0.001–100 nM). The cells were then incubated for 24 h at 37°C. In vitro re-epithelialization was documented by photography, and the amount ofmigration was quantitated by computer-assisted image analysis using NIHImage 1.6. The residual gap between the migrating keratinocytes wasmeasured and expressed as a percentage of the area of the scratch remainingunfilled. Each experiment was performed in triplicate.

Thymidine incorporation assay The proliferative potential of humankeratinocyte cultures with and without the presence of thalidomide wasdetermined by the thymidine incorporation assay of O’Keefe and Chiu(1988). Keratinocytes were plated in 24 well dishes (20,000 cells per cm2)and allowed to attach. Cells were then exposed to thalidomide diluted in Figure 1. Concentrations of thalidomide , 1 µM do not affect

keratinocyte viability. Keratinocytes were plated in 12 well plates,keratinocyte medium (0.001–100 nM) for 12 h and 2 µCi of (3H)thymidine(New England Nuclear Products, Boston, MA) were added per well. Cells allowed to attach and treated with increasing concentrations of thalidomide.

The medium was harvested and the amount of lactate dehydrogenasewere incubated at 37°C for 8 h. Medium was aspirated and the cells werewashed three times with ice-cold phosphate-buffered saline, twice with released into the medium calculated as described in the Materials and

Methods. Data are expressed as the mean 6 SD of triplicate values fromice-cold trichloracetic acid and solubilized with 0.1 M NaOH containing1% sodium dodecyl sulfate. Solubilized radioactivity was counted in a three experiments. *p , 0.05 compared with no added thalidomide.

** p , 0.001 compared with no added thalidomide.scintillation counter with Safety Solve. Ten wells were used for each

722 NASCA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

30. Therefore, the 8 µg per ml collagen matrix used in these Thalidomide does not alter keratinocyte gelatinasesecretion Many factors that promote keratinocyte motility alsoexperiments is a ‘‘suboptimal’’ matrix that provides migration indicesincrease MMP-9 activity, for example, hypoxia, collagen I, andof approximately 20. As shown in Fig 2, the presence of thalidomidecyclic adenosine monophosphate (Iwasaki et al, 1994; Sarret et al,at 0.01 nM, 0.1 nM, and 1 nM increased the migration indices1992a; O’Toole et al, 1997). MMP-9 is also known as type IVfrom 20.2 6 1.1 to 22.25 6 1.1 and 27.45 6 1.3, respectively.collagenase/gelatinase and has a molecular weight of 92 kDa. UsingTriplicate independent experiments were performed. Thalidomidestandard gelatin zymography, we examined the expression ofeffect on keratinocyte motility with suboptimal amounts of collagenMMP-9 in keratinocytes cultured with and without nontoxicwas statistically significant (p , 0.05), when analyzed by theconcentrations of thalidomide. Increasing concentrations of thalid-Student’s t test. On an ‘‘optimal’’ matrix of 15 µg per ml ofomide did not alter keratinocyte expression of MMP-9 (datacollagen, keratinocytes migrated maximally and no thalidomidenot shown).enhancement in cellular motility could be detected (data not shown).

To confirm the results of the colloidal gold assay, we assessedThalidomide increases keratinocyte IL-8 production IL-8motility in the scratch assay with and without thalidomide. In theserves as a keratinocyte autocrine factor and is a cytokine thatscratch assay, keratinocytes were plated densely in tissue culturepromotes keratinocyte motility. We wished to determine if thewells for 24 h incubated with mitomycin-C to inhibit proliferation,thalidomide-induced enhancement of keratinocyte motility wasthen a standardized scratch was made through the confluentassociated with an altered expression of keratinocyte-derived IL-8.monolayer, as previously described (Cha et al, 1996). KeratinocytesIL-8 production in the supernatant of keratinocytes with andfrom the cut edge of the scratch were then allowed to migrate for without thalidomide was measured by enzyme-linked immunosorb-12 h in the presence of increasing amounts of thalidomide and ent assay. Cell counts were performed to ensure that an approxi-photographed. The residual scratch area between inwardly migrating mately equal number of cells attached for each condition. IL-keratinocytes from the edges of the scratch was measured as 8 protein activity was detected in unconcentrated conditioneddescribed in Materials and Methods. As shown in Fig 3, the keratinocyte medium. As shown in Fig 5, the presence of thalidom-

keratinocytes migrated inwardly and covered a greater area of the ide increased IL-8 secretion in a concentration-dependent manner.scratch in the presence of thalidomide (compare 3B with 3G), Untreated keratinocytes, secreted basal levels of IL-8 (4.2 6 0.2 pgwhen compared with assays without thalidomide (3A). As shown per ml). In the presence of 0.1 nM, 1 nM, and 10 nM ofin the bar graph (Fig 3), keratinocytes on plastic exposed to thalidomide, the keratinocytes secreted 9.2 6 0.9 pg IL-8 per ml,thalidomide at concentrations of 0.001 nM, 0.01 nM, 0.1 nM, 11.5 6 0.8 pg IL-8 per ml, and 16.3 6 1 pg IL-8 per ml,1 nM, 10 nM, and 100 nM, migrated to fill 42 6 3.9, 52 6 4.8, respectively.61 6 5.9, 70 6 7.2, 82 6 7 and 90 6 9.2% of the scratch area,respectively. Concentrations greater than 0.1 nM significantly DISCUSSIONpromoted keratinocyte migration (p , 0.05, Student’s t test).

In recent years, thalidomide has been used successfully in thetreatment of several dermatoses characterized by loss of tissue,Thalidomide increases keratinocyte proliferation in vitro Inincluding aphthous stomatitis, genital aphthosis, Behcet’s diseaseorder to determine if thalidomide affects the growth potential(Grinspan et al, 1989; Revuz et al, 1990; Revuz, 1990; Gardner-of keratinocyte cultures, the ability of human keratinocytes toMedwin et al, 1994b), aphthous ulcers in AIDS patients (Patersonincorporate tritiated thymidine in the presence of noncytotoxicet al, 1995; Jacobson et al, 1997), and pyoderma gangrenosumdoses of thalidomide was assessed according to O’Keefe and Chiu(Munro and Cox, 1988; Rustin et al, 1990). Thalidomide has a(1988). As shown in Fig 4, human keratinocytes cultured in serum-number of side-effects but most of these are relatively uncommon.free medium in the presence of 0.01–100 nM of thalidomide hadTwo of the most serious side-effects are peripheral neuropathy,increased levels of thymidine incorporation compared with thosewhich is not uncommon and often dose-related, and its very highwithout thalidomide. This effect was concentration-dependent upincidence of teratogenicity in the event of fetal exposure.to a concentration of 0.1 nM. At concentrations . 0.1 nM,

To insure that the effects of thalidomide upon keratinocytethalidomide increased keratinocyte proliferation, but not as signi-migration and proliferation were not due to nonspecific toxicityficantly as with lower concentrations.of the drug, we determined the range at which thalidomidecould be used without causing nonspecific toxicity using a lactatedehydrogenase cytotoxicity assay. The noncytotoxic concentrationsof thalidomide used in the proliferation and motility assays werewell within the therapeutically effective plasma levels of thalidomideused in human diseases (Chen et al, 1989; Boughton et al, 1995).

The mode of action of thalidomide is unclear, but thalidomide isa potent immunomodulatory and anti-inflammatory agent (Patersonet al, 1995) that selectively reduces tumor necrosis factor-α expres-sion (Sampaio et al, 1991). Tumor necrosis factor-α promotesneutrophil maturation and keratinocyte IL-8 secretion, whichenhances neutrophil chemotaxis (Moreira et al, 1993; Costa et al,1994). Neutrophils at the wound site destroy contaminating bacteria(Tannesh et al, 1988), but this may cause further damage to healingtissue. Keratinocyte migration and proliferation are fundamentallyimportant steps in re-epithelializing skin wounds (Woodley et al,1993). In this study, thalidomide not only promoted keratinocytemotility in two independent migration assays, but also increasedthe cell’s proliferative potential. Thus, thalidomide may influencetwo fundamental mechanisms involved in wound healing. In thisstudy, low concentrations of thalidomide produced a marked

Figure 2. Thalidomide promotes keratinocyte migration in the increase in keratinocyte proliferation; however, higher concentra-colloidal gold assay. Keratinocytes were allowed to migrate for 16 h, intions were required to increase keratinocyte migration significantly.the presence of increasing concentrations of thalidomide. Ten migrationThis may be because keratinocyte migration requires new proteinindices were calculated for each condition and the experiment was repeatedsynthesis, redistribution of lamellipodia-associated proteins, andthree times. Error bars; mean 6 SD. *p , 0.004 compared with no added

thalidomide. synthesis and secretion of collagenases (O’Toole et al, 1997). These

VOL. 113, NO. 5 NOVEMBER 1999 THALIDOMIDE AND KERATINOCYTES 723

Figure 3. Thalidomide promotes keratinocyte migration in the in vitro scratch assay. An in vitro wound was introduced in confluent cultures ofnormal human keratinocytes, as described in the Materials and Methods. The cells were incubated without thalidomide (A) or with increasing concentrationsof thalidomide (B–G). After 24 h, the cells were photographed under phase-contrast microscopy. Scale bar: 250 µm. The percentage of the original scratcharea remaining unfilled was calculated by image analysis and expressed as the mean 6 SD of three observations (graph). 10–12–10–7 5 0.001–100 nM.

Figure 5. Thalidomide increases human keratinocyte IL-8Figure 4. Thalidomide promotes keratinocyte proliferation in vitro. secretion. Keratinocytes cultured in basal medium were plated on plasticSubconfluent keratinocytes in 24 well plates were exposed to increasing at a concentration of 15,000 cells per cm2. After cell attachment, increasingconcentrations of thalidomide for 12 h, then incubated with 2 µCi of concentrations of thalidomide were added to the dishes. After 12 h,thymidine for an additional 8 h. Cellular samples were prepared as described the concentration of IL-8 was measured in unconcentrated conditionedin the Materials and Methods and the amount of solubilized radioactivity medium. Error bars, mean 6 SD of three experiments performed inwas counted in a scintillation counter. Error bars, mean 6 SD of three triplicate. *p , 0.001 compared with control without thalidomide.experiments performed in triplicate. ** p , 0.0005 compared with control ** p , 0.0005 compared with control without thalidomide.without thalidomide. *p , 0.001 compared with control withoutthalidomide.

IL-8 is known to enhance human keratinocyte chemotaxis(Schroder, 1992) and proliferation (Tuschil et al, 1992) and to beprocesses may require higher doses of thalidomide compared with

DNA synthesis. increased in diseases characterized by epidermal hyperproliferation,such as psoriasis (Nickoloff et al, 1991, 1994). In this study, lowMatrix-induced promotion of keratinocyte motility is associated

with increased synthesis of collagenases (Petersen et al, 1987, 1989; concentrations of thalidomide (0.01–0.1 nM) promoted humankeratinocyte proliferation, whereas higher concentrations also pro-Sarret et al, 1992a; Iwasaki et al, 1994). Degradation of matrix

allows for detachment of keratinocyte-matrix adhesions, and this moted human keratinocyte proliferation, but not as markedly asthe lower concentrations. These data are in accordance withprocess allows the cell to continue to migrate during re-epithelializ-

ation. Our study demonstrates that thalidomide does not alter the other studies of other cell types showing opposite biologic effectsdepending upon the concentration of thalidomide used (McHughexpression of keratinocyte-derived MMP-9, a collagenase linked

to keratinocyte migration on a collagen matrix (Sarret et al, 1992a). et al, 1995). IL-8 secretion, evaluated by enzyme-linked immuno-sorbent assay, showed a 2–4-fold increase compared with controlTherefore, the enhancement of human keratinocyte motility by

thalidomide likely involves some other mechanism. human keratinocytes in the presence of thalidomide. Therefore,

724 NASCA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Ewan PW: The immunosuppressive drug thalidomide induces T-helper cell typethe thalidomide-induced enhancement of keratinocyte motility2 (Th2) and concomitantly inhibits Th1 cytokine production in mitogen- andmay be mediated via an increased expression of IL-8 which would antigen-stimulated human peripheral blood mononuclear cell cultures. Clin Exp

act as a pro-migratory autocrine factor. Immunol 99:160–167, 1995Moncada B, Baranda ML, Gonzalez-Amaro R, Urbina R, Loredo CE: ThalidomideKeratinocyte migration and proliferation are independent mech-

effect on T cell subsets as a possible mechanism of action. Int J Lepr 53:201–anisms (Sarret et al, 1992b; Woodley et al, 1993; Iwasaki et al,205, 19851994). The timing of the proliferative and locomotion effects during Moreira AL, Sampaio EP, Zmuidzinas A, Frindt P, Smith KA, Kaplan G: Thalidomide

wound healing are different, as the enhancement of keratinocyte exerts its inhibitory action on tumor necrosis factor a by enhancing mRNAdegradation. J Exp Med 177:1675–1680, 1993migration occurs early, whereas the increase in cell division takes

Munro CS, Cox NH: Pyoderma gangrenosum associated with Behcet’s syndrome-place later (Woodley et al, 1993). Moreover, keratinocyte prolifera-response to thalidomide. Clin Exp Dermatol 13:408–410, 1988tion may be enhanced by soluble factors and cytokines that have Naafs B, Faber WR: Thalidomide therapy. An open trial. Int J Dermatol 24:131–

no effect, or, alternatively, have an inhibitory action on keratinocyte 134, 1985Nasca MR, Micali G, West DP: Thalidomide—a review. in press, 1999motility (Sarret et al, 1992b). Epidermal growth factor and trans-Neubert R, Nogueira AC, Neubert D: Thalidomide and the immune system. 2.forming growth factor-α enhance both keratinocyte migration and

Changes in receptors on blood cells of a healthy volunteer. Life Sci 51:2107–proliferation (Cha et al, 1996), but these effects take place by 2116, 1992independent mechanisms (Chen et al, 1993). In this study, we Neubert R, Nogueira AC, Neubert D: Thalidomide derivatives and the immune

system. I. Changes in the pattern of integrin receptors and other surface markersfind that thalidomide enhances both keratinocyte migration andon T lymphocyte subpopulations of marmoset blood. Arch Toxicol 67:1–17, 1993proliferation in vitro. The therapeutic value of thalidomide in the

Neubert R, Hinz N, Thiel R, Neubert D: Down-regulation of adhesion receptors ontreatment of chronic wounds has yet to be determined. The positive cells of primate embryos as a probable mechanism of the teratogenic action ofeffect of thalidomide on both migration and proliferation in vitro, thalidomide. Life Sci 58:295–316, 1996

Nickoloff BJ, Karabin GD, Barker JN, et al: Cellular localization of interleukin-8and the many anecdotal reports of successful treatment of chronicand its inducer, tumor necrosis factor-alpha in psoriasis. Am J Pathol 138:129–ulcerating conditions with thalidomide suggest that it may be of140, 1991value in conditions where there is deficient re-epithelialization. Nickoloff BJ, Mitra RS, Varani J, Dixit VM, Polverini PJ: Aberrant production ofinterleukin-8 and thrombospondin-1 by psoriatic keratinocytes mediatesangiogenesis. Am J Pathol 144:820–828, 1994

Nogueira AC, Neubert R, Helge H, Neubert D: Thalidomide and the immune system.3. Simultaneous up- and down-regulation of different integrin receptors onThis study was supported by Northwestern Memorial Foundation (DPW), ahuman white blood cells. Life Sci 55:77–92, 1994Howard Hughes Medical Institute Physician Fellowship (EOT), and Grants P01

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