- Email: [email protected]
Thalidomide suppressed interleukin-6 but not tumor necrosis factor-alpha in volunteers with experimental endotoxemia
ORIGINAL ARTICLES Thalidomide suppressed interleukin-6 but not tumor necrosis factor-alpha in volunteers with experimental endotoxemia EDWARD SHANNON, ROBERT NOVECK, FELIPE SANDOVAL, BURDE KAMATH, and MICHAEL KEARNEY BATON ROUGE, NEW ORLEANS, AND KENNER, LA
An early rationale for using thalidomide to treat erythema nodosum leprosum had been based on some reports that it suppresses tumor necrosis factor-alpha (TNF-␣). However, in vivo and in vitro studies have yielded variable results, having shown that thalidomide can either enhance or suppress TNF-␣. Since the course of circulating cytokines like TNF-␣ after infusion of endotoxin into volunteers is reproducible and characteristic, we investigated the effect of thalidomide on endotoxin-induced synthesis of TNF-␣, interleukin (IL)-6, and IL-8. The cytokine response from 18 placebo-treated subjects who had undergone the endotoxin challenge were pooled with a placebo-treated subject from the current study and were compared with 4 subjects who received thalidomide (100 mg) every 6 h for 5 doses before endotoxin challenge. Thirty minutes after the last dose of thalidomide or placebo, volunteers were infused with 4-ng/kg endotoxin. Plasma was collected and assayed for cytokines by enzyme-linked immunosorbent assay. Endotoxin evoked the synthesis of the cytokines in all volunteers. The peak response for TNF-␣ was 1.5 h, 2.5 h for IL-8, and 3.0 h for IL-6. Thalidomide did not significantly delay the release of cytokines into the circulating blood. At the peak response, thalidomide reduced the concentration of the cytokines in the plasma. Using the area under the dose response curve (AUC0 to 24 h), thalidomide reduced the AUC for IL-6 by 56%, for IL-8 by 30%, and TNF-␣ by 32%. In this model, thalidomide did not suppress TNF-␣ or IL-8, but it did suppress IL-6 at 4-h postinfusion with lipopolysaccharide (P ⴝ 0.004), at 6 h (P ⴝ 0.014), at 12 h (P ⴝ 0.001), and at 16 h (P ⴝ 0.012). (Translational Research 2007;150: 275–280) Abbreviations: AUC ⫽ area under the curve; ELISA ⫽ enzyme-linked immunosorbent assay; ENL ⫽ erythema nodosum leprosum; FDA ⫽ U.S. Food and Drug Administration; HUVEC ⫽ human umbilical vein endothelial cell; IFN-␥ ⫽ interferon-gamma; IL ⫽ interleukin; LPS ⫽ lipopolysaccharide; TNF-␣ ⫽ tumor necrosis factor-alpha
From the National Hansen’s Disease Programs, Baton Rouge, LA; the Clinical Research Center/MDS Pharma Services, New Orleans, LA; the Reliant Laboratory Services, Kenner, LA; and the Department of Pathobiological Sciences, Louisiana State University, Baton Rouge, LA. Submitted for publication February 23, 2007; revision submitted May 21, 2007; accepted for publication May 23, 2007.
Reprint requests: E. J. Shannon, Lab Research, Louisiana State University, SVM, Skip Bertman Dr., Baton Rouge, LA 70803; E-mail: [email protected]. 1931-5244/$ – see front matter © 2007 Mosby, Inc. All rights reserved. doi:10.1016/j.trsl.2007.05.003
275
276
Translational Research November 2007
Shannon et al
Thalidomide possesses anti-inflammatory and immunomodulatory properties.1 Thalidomide is approved by the U.S. Food and Drug Administration (FDA) for the treatment of the cutaneous manifestations of moderateto-severe erythema nodosum leprosum (ENL), and for the treatment of newly diagnosed cases of multiple myeloma in combination with dexamethasone. It is being evaluated for efficacy in the treatment of a variety of conditions such as autoimmune diseases, solid tumor, and other blood cancers. The rationale for its use varies with the condition under treatment. In diseases where the pathology is associated with tumor necrosis factor-alpha (TNF-␣), thalidomide is used for its putative ability to downregulate TNF-␣. Thalidomide can regulate the synthesis of TNF-␣, but it does so in a confusing and contradictory manner. The inflammatory condition most successfully treated with thalidomide is ENL. After treatment of ENL with thalidomide, there are reports of significant decrease in TNF-␣,2 no change in TNF-␣,3 or a marginal increase in TNF-␣.4,5 In the treatment of inflammatory aphthous ulcers, dosing with thalidomide was associated with an increase in TNF-␣;6 and in a trial using thalidomide to treat toxic epidermal necrolysis, patients treated with thalidomide were removed from the trial caused by increased mortality possibly related to increased TNF-␣ plasma concentrations.7 Using human blood-derived monocytes stimulated with endotoxin to evoke the synthesis of TNF-␣, the results vary. One study described thalidomide as having a suppressive effect,8 whereas we identified an enhancing effect.4 The results also differ when rodents are treated with thalidomide and challenged with endotoxin to induce septic shock and death. Some studies showed thalidomide to be protective.9,10 Others reported no significant effect of thalidomide on the TNF-␣ response after lipopolysaccharide (LPS)-induced shock.11,12 The different effects of thalidomide on TNF-␣ from human clinical trials and from sepsis studies in rodents may be caused by many variables. Some of these variables may involve the inflammatory condition treated. In the rodent studies factors included, the route of administration [intraperitoneal vs gavage], dose of thalidomide [6 –300 mg/kg], and the timing in which thalidomide was administrated in relation to the induction of shock [either before or after challenge with endotoxin]. Consequently, interpretation of the literature regarding the ability of thalidomide to consistently downregulate TNF-␣ in inflammatory conditions is confusing. Since the detection of cytokines such as TNF-␣ into the circulating blood volume in endotoxin models is reproducible regardless of species studied (human, primate, pig, rat, or mouse),13 we investigated the effect of
thalidomide on endotoxin-invoked synthesis of TNF-␣, interleukin (IL)-6, and IL-8 in healthy male volunteers. METHODS Subjects. Both clinical protocols were approved by an independent investigational review board. In addition, for the study involving thalidomide, the protocol was approved by the Division of Anti-infective Drug Products of the FDA and by the Hansen’s Disease Programs Research Committee. All participants in the clinical trials gave written informed consent before undertaking any study-related procedures. All subjects participating in the thalidomide trial in February 2003 were counseled on the reproduction risks associated with thalidomide and viewed a patient education video on the risks of fetal exposure associated with Thalomid® provided by Celgene Corporation (Summit, NJ). All subjects had agreed not to father a child and to wear a condom for any sexual intercourse for 1 month afterward. The originally planned, double-blinded, placebo-controlled clinical trial was permanently interrupted by severe weather (Hurricane Katrina), so it was decided to pool placebo data from a separate double-blind study performed 2 years later under identical conditions. See the Statistical Methodology section for the method used to add these placebo data. The added 18 placebo subjects did not view the video, but they did receive the 4.0-ng/kg dose of LPS that was prepared from the same lot of LPS used in the thalidomide-treated study. Endotoxin. Purified LPS (Clinical Center Reference Endotoxin, lot 2) 10,000 endotoxin units [1000 ng] per vial, obtained from the Pharmacy Development Service, Clinical Center, National Institutes of Health, Bethesda, Md, was diluted in sterile pyrogen-free water to 200 ng/mL. The LPS was administered as a bolus and at a dose of 4.0 ng/kg of body weight [the maximum dose allowed for human volunteers]. Thalomid® (thalidomide). One hundred-mg capsules and identical matching placebo capsules were kindly provided by Celgene Corporation. Thirty minutes after the last dose (100 mg p.o. q 6 h ⫻ 5 doses), the volunteers were infused with endotoxin. Study design. The protocol was a double-blind, placebocontrolled, single-site study. All subjects were confined to the research facility, beginning on the evening before study initiation and concluding 24 h after receiving endotoxin. The study was conducted in full conformance with the principles of the “Declaration of Helsinki” and its amendments, or with the laws and regulations of the locality in which the research was conducted, whichever afforded the greater protection to the individual. Assessments. Blood was collected in tubes containing sodium heparin. Before infusion of endotoxin, the plasma was collected before ingestion of thalidomide and 30 min after the last dose (0 h). After infusion of LPS, plasma was collected at 0.5, 1, 1.5, 2.5, 4,5, 6,5, 8.5, 12.5, 16.5, and 24 h and on the 7th and 30th day. For the assessment of thalidomide in the blood, the plasma was acidified using 25 mmol/L (pH 1.5) citrate buffer.14 The concentration of thalidomide was determined by high-pressure liquid chromatography using a modification of the method of Eriksson et al.15 Before and after
Translational Research Volume 150, Number 5
the infusion of endotoxin, plasma for cytokine analysis was collected at 0.0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 16, and 24 h and was frozen and analyzed by enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s procedure (Pierce Endogen Rockford, Ill). The concentrations of the cytokines were determined using software KC4V 3.3 (BioTek, Instruments, Inc., Highland Park Winooski, Vt). The areas under the curve (AUCs) were calculated using GraphPad Prism v 4.0 (San Diego Calif). Assessment of lymphocyte subsets and IL-2 and interferon-gamma (IFN-␥). Before and shortly after receiv-
ing thalidomide or placebo, blood was collected in tubes containing EDTA. The cells expressing CD3⫹/CD4⫹; CD3⫹/CD8⫹ and CD3⫹/CD69⫹ molecules was determined by using BD-conjugated MoAbs and flow cytometry analysis on a BD FACScan instrument (San Jose, Calif). Flow cytometry was performed using directly conjugated MoAbs in double-labeling and triple-labeling experiments. Appropriate fluorochromes directly-conjugated to mouse immunoglobulins were used as isotope-matched negative controls. IL-2 and IFN-␥ were assayed by ELISA kits from Pierce Endogen. Statistical methodology. The SAS statistical package (version 9.1.3; The SAS Institute, Inc., Cary, NC) was used to analyze the loge-transformed data in a repeated measures design as a split-plot arrangement of treatments with TREATMENT and SUBJECT (TREATMENT) as main plot effects. TIME and TREATMENT*TIME interactions were included on the subplot. When overall significance (P ⱕ ⫺0.05) was detected, post hoc pair-wise comparisons were performed with the Tukey HSD test for main effect comparisons and t tests of least-squares means for comparisons of interaction levels. Distributional statistics (Shapiro-Wilk test of normality, stem-leaf diagrams, box-whisker plots, and examination of individual extreme values) performed on the placebo group at each time point justified the inclusion of the additional cohort of 18 subjects into the analysis for a final placebo group of 19 subjects.
RESULTS
Thalidomide was well tolerated, and no adverse events occurred before administration of endotoxin. After the last dose of thalidomide, the mean plasma concentration of thalidomide was 1.6 g/mL. The concentrations ranged from 1.0 to 2.5 g/mL. Thalidomide remained in 3 of 4 volunteers 16.5 h after infusion with LPS at a mean concentration of (0.4 ⫾ 0.32 g/mL). After 24 h, 2 subjects had thalidomide detected at 0.34 and 0.35 g/mL. No thalidomide was detected at day 7 or day 30. The demographic characteristics of the volunteers infused with LPS are summarized in Table I. LPS evoked the synthesis of the cytokines in all volunteers (Fig 1, A–C). TNF-␣ peaked at 1.5 h after the infusion of endotoxin. This was 1 h before the peak responses for IL-8 (2.5 h) and 1.5 h before the peak response for IL-6 (3.0 h).
Shannon et al
277
Table I. The demographic characteristics of the volunteers infused with LPS A. Volunteers infused with 4 ng/kg LPS (n ⫽ 23) 1. Signed Informed Consent 2. 18–45 years of age, 65–80 kg 3. Nonsmokers, no recent history of any conditions that could affect an LPS response 4. Did not donate blood within the previous 2 months. 5. Did not receive LPS or participated in an investigational trial within the previous 30 days 6. Had a normal stable body temperature: average of 3 consecutive oral temperatures between 97.0oF and 98.8oF [Not to differ by ⬎0.4oF before challenge with LPS] B. Volunteers treated with thalidomide and infused with 4 ng/ kg LPS (n ⫽ 4) 1. Age (y) 21–43 2. Height (cm) 165–191 3. Weight (kg) 63.8–93.6
The cytokine response to LPS in the volunteers is summarized in Table II. Compared with the placebo-treated group, thalidomide did not significantly delay the appearance of the peak response of the cytokines (Fig 1, A–C). In comparing the area under the dose response curve from the 0 h to 24 h (AUC0-24) for the placebo-treated group to the thalidomide-treated group, thalidomide reduced the AUC0-24in the following order: IL-6 ⬎ TNF-␣ ⬎ IL-8 (Table II). In agreement with a previous study, IL-6 showed considerable individual variability, a peak response 2 h after a bolus injection of LPS, and a decrease that returned to baseline after 8 h.16 To reduce the variance within the groups, we used the natural log (to the base e) to transform the data. Comparing the placebo group (n ⫽ 19) to the thalidomide-treated group (n ⫽ 4), significant suppression of IL-6 was observed at 4 h (P ⫽ 0.0043), 6 h (P ⫽ 0.0145), 12 h (P ⫽ 0.0014), and 16 h (P ⫽ 0.012) (Fig 1, B). No statistically significant suppression of TNF-␣ was detected (Fig 1). It is interesting that there seemed to be a shift in IL-8. After the peak response IL-8 decreased in the thalidomidetreated group, but at 12 h, there was a shift in activity. From the 12-h to 24-h sampling of the plasma, there seemed to be an increase in IL-8 in the thalidomidetreated group. DISCUSSION
The primary objective of this study was to determine whether thalidomide could attenuate the TNF-␣ response evoked by endotoxin. Thalidomide was given before challenge with endotoxin and at doses recommended to treat acute ENL.17 Thalidomide was well tolerated, and no adverse events occurred before administration of endotoxin.
278
Shannon et al
Fig 1. Inflammatory molecules. Mean (⫾ SE) of loge -transformed data of the pg/mL of TNF-␣ (1a), IL-6 (1b), and IL-8 (1c) detected in plasma after infusion of 4.0 ng/kg of endotoxin (t ⫽ 0 h). Administration of endotoxin was preceded by ingestion of thalidomide (100 mg every 6 h ⫻ 5 doses; n ⫽ 4) [—] or 0 mg in the placebo n ⫽ 19 [- - -].
Using fluorescence microscopy, we reported that thalidomide decreased the absolute numbers of CD4⫹ cells in healthy males18 and in patients with ENL.19 In this study using flow cytometry analysis, thalidomide
Translational Research November 2007
did not significantly change the number of white blood cell/L, % lymphocytes, % granulocytes, and % monocytes, nor did it significantly alter the percent or absolute number of CD3⫹/CD4⫹; CD3⫹/CD8⫹ and CD3⫹/CD69⫹ lymphocytes (paired t test, data not shown). Thalidomide in vitro can increase mitogenstimulated mononuclear cell synthesis of IL-2 in healthy males20 and HIV-infected males.21 In this study, before and after the ingestion of thalidomide and before infusion of LPS, plasma levels of IL-2 and IFN-␥ were below the pg/mL detection levels of the standard curve on the ELISA plate (paired t test, data not shown). LPS evoked the synthesis of the cytokines in all volunteers. The appearance of TNF-␣ preceded the appearance of IL-6 and IL-8. This is in accordance with similar studies using this model.22 In our study, the cytokines at their peak responses that were most significantly suppressed by thalidomide were IL-6 and IL-8. IL-8 is a chemokine and a potent chemotaxin for neutrophils, which is the predominant cell type found in ENL skin lesions. It is produced by endothelial cells to facilitate leukocyte emigration. Dunzenforfer et al23 showed that upon stimulation with TNF-␣ or endotoxin, human umbilical vein endothelial cells (HUVECs) produce IL-8. Thalidomide, at concentrations similar to that in the plasma of the volunteers in our study, seems to have affected this process in a bidirectional manner. That is, the synthesis of IL-8 by HUVEC cells was augmented when they were stimulated with TNF-␣ and inhibited when the cells were stimulated with endotoxin. This bidirectional effect by thalidomide in this in vitro model may help explain the suppressive/enhancing effect of thalidomide on IL-8 in our in vivo study. In the endotoxin challenge model, endotoxin is cleared from the blood within 30 min after intravenous administration,24 and the major source of TNF is reported to be the liver.25 Therefore it is possible that, early in the response, endotoxin inhibited endothelial cell synthesis of IL-8, whereas later in the response TNF-␣ facilitated the synthesis of IL-8 by endothelial cells. An ability of thalidomide to regulate IL-8 secreted by endothelial cells could offer a partial explanation of its effectiveness in ENL. Recently, thalidomide has been granted FDA approval for treatment, in combination with dexamethasone, of newly diagnosed cases of multiple myeloma. The approval is based on an accelerated response rate compared with dexamethasone alone. Corticosteroids and estrogens are potent inhibitors of IL-6 production as a result of their inhibition of IL-6 gene transcription.26 It is interesting to speculate that Thalomid® (thalidomide) may synergize with dexamethasone and
Translational Research Volume 150, Number 5
Shannon et al
279
Table II. Detection of TNF-␣, IL-6, and IL-8 in the plasma of 19 placebo-treated and 4 thalidomide-treated volunteers after infusion of 4.0 ng/kg of endotoxin Cmax in pg/mL (tmax)
TNF-␣ IL-6 IL-8
AUC pg.hr/mL
% Suppression
Placebo
Thalidomide
Placebo
Thalidomide
AUC0-24
2048 (1.5 h) 2263 (3.0 h) 2338 (2.5 h)
1184 (1.5 h) 1089 (3.0 h) 1484 (2.5 h)
3370 7701 8901
2314 3378 6231
32 56 30
Abbreviations: AUC0-24, Area under the curve [(pg)(hr(0-24)/mL)] values calculated by trapezodial method; Cmax, Maximum concentration (pg/mL) for TNF-␣, IL-6, and IL – 8; % Suppression, [1 – (Mean AUC0-24 for thalidomide/(Mean AUC0-24 for placebo)] ⫻ 100; Tmax, Time in hours (0-24) to achieve maximum concentration.
markedly suppress the synthesis and/or release of IL-6. In a similar endotoxin challenge model using human volunteers, IL-6, IL-8, and TNF-␣ were significantly suppressed, in a dose response manner, by prednisolone.27 IL-6 has multiple activities, including promotion of B-cell growth and secretion of immunoglobulins by B cells, and stimulating the growth of plamacytomas, myelomas, and several B lymphomas.28 Among leprosy patients, we have shown that thalidomide can decrease IgM antibody synthesis and have speculated that this may offer an explanation for its ability to suppress an antibody-mediated Arthus-like ENL reaction.29 It seems that ENL is a hypersensitivity reaction to M. leprae having humoral and cell- mediated immune components. The roles played by antibody, complement, and sensitized T cells in precipitating ENL have yet to be elucidated. The pathogenesis of ENL is likely to be the outcome of a complex interaction of immunologic and nonspecific anti-inflammatory events. Evidence accumulating is that TNF-␣ is not the sole cytokine targeted by thalidomide in ENL and other inflammatory conditions. Recently, IL-6 but not TNF-␣ was shown to be decreased in patients treated for ENL with thalidomide.3 With the following considerations: (1) that the originally planned clinical trail was permanently interrupted by severe weather, (2) that most of the placebo data are from a separate study performed under identical conditions, and (3) that infusion of LPS mimics the ENL inflammatory response; this study adds to the evidence that downregulation of IL-6 may be a site of action of thalidomide in ENL. REFERENCES
1. Zwingerberger K, Wnendt S. Immunomodulation by thalidomide: systematic review of the literature of unpublished observations. J Inflamm 1996;46:177–211. 2. Sampaio E, Kaplan G, Miranda A, Nery JAC, Miguel CP, Viana, S, et al. The influence of thalidomide on clinical leprosum. J Infect Dis 1993;168:408 –14.
3. Villahermosa LG, Fajardo T, Abalos, RM, Balagona MV, Tan EV, Cellona RV, et al. A randomized, double-blind, doubledummy, controlled dose comparison of thalidomide for treatment of erythema nodosum leprosum. Am J Trop Med Hyg 2005;72: 518 –26. 4. Shannon EJ, Sandoval FG. Thalidomide can be either agonistic or antagonistic to LPS evoked synthesis of TNF-alpha by mononuclear cells. Immunopharmacol Immunotoxicol 1996;18:59 –72. 5. Haslett P, Roche P, Butlin C, Macdonald M, Shrestha N, Manandhar R, et al. Effective treatment of erythema nodosum leprosum with thalidomide is associated with immune stimulation. J Infect Dis 2005;192:2045–53. 6. Jacobson J, Greenspan J, Spritzler J, Ketter N, Fahey J, Jackson J, et al. Thalidomide for the treatment of oral aphthous ulcers in patients with immunodeficiency virus infection. New Engl J Med 1997;336:1487–93. 7. Wolkenstein P, Latarjet J, Roujeau, J, Duguet C, Boudeau S, Vaillant, L, et al. Randomized comparison of thalidomide versus placebo in toxic epidermal necrolysis. Lancet 1998;352:1586 – 89. 8. Sampaio E, Sarno E, Gailly R, Cohn Z, Kaplan G. Thalidomide selectively inhibits tumor necrosis factor ␣ production by stimulated human monocytes. J Exp Med 1991;173:699 –703. 9. Schmidt H, Rush B, Simonian G, Murphy T, Hsieh J, Condon M. Thalidomide inhibits TNF response and increases survival following endotoxin injection in rats. J Surg Res 1996;3:142– 6. 10. Moreira A, Wang J, Sarno E, Kapan G. Thalidomide protects mice against LPS-induced shock. Braz J Med Bio Res 1997;30: 1199 –207. 11. Ishikawa M, Kanno S, Takayangi M, Takayangi Y, Sasaki K, Thalidomide promotes the release of tumor necrosis factor-␣ (TNF-␣) and lethality by lipopolysaccharide in mice. Bio Phar Bull 1998;21:638 – 40. 12. Fernandez-Martinez E, Morales-Rios MS, Perez-Alverez V, Muriel P. Immunomodulatory effects of thalidomide analogs on LPS-induced plasma and hepatic cytokines in the rat. Biochem Pharmacol 2004;68:1321–9. 13. Hack CE, Aarden LA, Thijs LG. Role of cytokines in sepsis. Adv Immunol 1997;66:101–95. ¨ , Ekberg H. Determination of 14. Eriksson T , Björkman S, Fyge A thalidomide in plasma by high-performance liquid chromatography: avoiding hydrolytic degradation. J Chromatogr 1992;582: 211– 6. ¨ , Höiglund P. Sterospe15. Eriksson T, Björkman S, Bodil R, Fyge A cific determination, chiral inversion in vitro and pharmacokinetics in humans of the enanthiomers of thalidomide. Chirality 1995;7:44 –52. 16. Endler G, Marsik C, Joukhadar C, Marculescu R, Mayr F, Mannhalter C, et al. The interleukin-6 G(-174) C promoter
280
17. 18.
19.
20.
21.
22.
Shannon et al
polymorphism does not determine plasma interleukin-6 concentrations in experimental endotoxemia in humans. Clin Chem 2004;50:195–200. Hastings RC Leprosy. 137 1993194 Churchill Livingstone Edinburgh, UK 155. Gad SM, Shannon EJ, Krotoski WA, Hastings RC. Thalidomide induces imbalances in T-lymphocyte sub-populations in the circulating blood of healthy males. Lepr Rev 1985;56:35–9. Shannon EJ, Mestawat E, Haile-Mariam HS, Tebebe YB, Tadesse G. Thalidomide’s effectiveness in erythema nodosum leprosum is associated with a decrease in CD4⫹cells in peripheral blood. Lepr Rev 1992;63:5–11. Shannon EJ, Sandoval F. Thalidomide increases the synthesis of IL-2 in cultures of human mononuclear cells stimulated with Concanavalin-A, staphylococcal entertoxin A, and purified protein derivative. Immunopharmacology 1999;31:109 –16. Shannon EJ, Asseffa A, Pankey G, Sandoval F, Lutz B. Thalidomide’s ability to augment the synthesis of IL-2 in vitro in HIV-infected patients is associated with the percentage of CD4⫹ cells in their blood. Immunopharmacology 2000;46:175–9. Suffredini AF, Reda D, Banks SM, Tropea M, Agosti LM, Miller R. Effects of recombinant dimeric TNF receptor on human inflammatory responses following intravenous endotoxin administration. J Immunol 1995;155:5038 – 45.
Translational Research November 2007
23. Dunzenforfer S, Manfred H, Weidermann C. Inducer-specific bidirectional regulation of endothelial interleukin-8 produced by thalidomide. Immunopharmacology 1999;43:59 – 64. 24. von der Moehlen MAM, Kimmings AN, Wedel NI, Mevissen ML, Jasen J, Friedmann N, et al. Inhibition of endotoxin-induced cytokine release and neutrophil activation in humans by use of recombinant bactericidal/permeability-increasing protein. J Infec Dis 1995;172:144 –51. 25. Fong YM , Marano MA, Mokdawer LL, Wei H, Calvano SE, Denney JS, et al. The acute spanchnic and peripheral tissue metabolic response to endotoxin in humans. J Clin Invest 1990;85:1896 – 1904. 26. Hasan SI, Blaney B, Turk JL. Effect of anticancer drugs on the release of interleukin-6 in vitro. Cancer Immunol Immunother 1992;34:228 –32. 27. de Kruif MD, Lemaire LC, Giebelen IA, van Zoelen MAD, Pater JM, van den Pangaart PS, et al. Prednisolone dose-dependently influences inflammation and coagulation during human endotoxemia. J Immunol 2007;178:1845–51. 28. Akira S, Taga T, Kishimoto. Interleukin-6 biology and medicine. Adv Immunol 1993;54:1–78. 29. Shannon EJ, Miranda RO, Morales MJ, Hastings RC. Inhibition of de novo antibody synthesis by thalidomide as a relevant mechanism of action in leprosy. Scand J Immunol 1981;13:17–25.
An early rationale for using thalidomide to treat erythema nodosum leprosum had been based on some reports that it suppresses tumor necrosis factor-alpha (TNF-␣). However, in vivo and in vitro studies have yielded variable results, having shown that thalidomide can either enhance or suppress TNF-␣. Since the course of circulating cytokines like TNF-␣ after infusion of endotoxin into volunteers is reproducible and characteristic, we investigated the effect of thalidomide on endotoxin-induced synthesis of TNF-␣, interleukin (IL)-6, and IL-8. The cytokine response from 18 placebo-treated subjects who had undergone the endotoxin challenge were pooled with a placebo-treated subject from the current study and were compared with 4 subjects who received thalidomide (100 mg) every 6 h for 5 doses before endotoxin challenge. Thirty minutes after the last dose of thalidomide or placebo, volunteers were infused with 4-ng/kg endotoxin. Plasma was collected and assayed for cytokines by enzyme-linked immunosorbent assay. Endotoxin evoked the synthesis of the cytokines in all volunteers. The peak response for TNF-␣ was 1.5 h, 2.5 h for IL-8, and 3.0 h for IL-6. Thalidomide did not significantly delay the release of cytokines into the circulating blood. At the peak response, thalidomide reduced the concentration of the cytokines in the plasma. Using the area under the dose response curve (AUC0 to 24 h), thalidomide reduced the AUC for IL-6 by 56%, for IL-8 by 30%, and TNF-␣ by 32%. In this model, thalidomide did not suppress TNF-␣ or IL-8, but it did suppress IL-6 at 4-h postinfusion with lipopolysaccharide (P ⴝ 0.004), at 6 h (P ⴝ 0.014), at 12 h (P ⴝ 0.001), and at 16 h (P ⴝ 0.012). (Translational Research 2007;150: 275–280) Abbreviations: AUC ⫽ area under the curve; ELISA ⫽ enzyme-linked immunosorbent assay; ENL ⫽ erythema nodosum leprosum; FDA ⫽ U.S. Food and Drug Administration; HUVEC ⫽ human umbilical vein endothelial cell; IFN-␥ ⫽ interferon-gamma; IL ⫽ interleukin; LPS ⫽ lipopolysaccharide; TNF-␣ ⫽ tumor necrosis factor-alpha
From the National Hansen’s Disease Programs, Baton Rouge, LA; the Clinical Research Center/MDS Pharma Services, New Orleans, LA; the Reliant Laboratory Services, Kenner, LA; and the Department of Pathobiological Sciences, Louisiana State University, Baton Rouge, LA. Submitted for publication February 23, 2007; revision submitted May 21, 2007; accepted for publication May 23, 2007.
Reprint requests: E. J. Shannon, Lab Research, Louisiana State University, SVM, Skip Bertman Dr., Baton Rouge, LA 70803; E-mail: [email protected]. 1931-5244/$ – see front matter © 2007 Mosby, Inc. All rights reserved. doi:10.1016/j.trsl.2007.05.003
275
276
Translational Research November 2007
Shannon et al
Thalidomide possesses anti-inflammatory and immunomodulatory properties.1 Thalidomide is approved by the U.S. Food and Drug Administration (FDA) for the treatment of the cutaneous manifestations of moderateto-severe erythema nodosum leprosum (ENL), and for the treatment of newly diagnosed cases of multiple myeloma in combination with dexamethasone. It is being evaluated for efficacy in the treatment of a variety of conditions such as autoimmune diseases, solid tumor, and other blood cancers. The rationale for its use varies with the condition under treatment. In diseases where the pathology is associated with tumor necrosis factor-alpha (TNF-␣), thalidomide is used for its putative ability to downregulate TNF-␣. Thalidomide can regulate the synthesis of TNF-␣, but it does so in a confusing and contradictory manner. The inflammatory condition most successfully treated with thalidomide is ENL. After treatment of ENL with thalidomide, there are reports of significant decrease in TNF-␣,2 no change in TNF-␣,3 or a marginal increase in TNF-␣.4,5 In the treatment of inflammatory aphthous ulcers, dosing with thalidomide was associated with an increase in TNF-␣;6 and in a trial using thalidomide to treat toxic epidermal necrolysis, patients treated with thalidomide were removed from the trial caused by increased mortality possibly related to increased TNF-␣ plasma concentrations.7 Using human blood-derived monocytes stimulated with endotoxin to evoke the synthesis of TNF-␣, the results vary. One study described thalidomide as having a suppressive effect,8 whereas we identified an enhancing effect.4 The results also differ when rodents are treated with thalidomide and challenged with endotoxin to induce septic shock and death. Some studies showed thalidomide to be protective.9,10 Others reported no significant effect of thalidomide on the TNF-␣ response after lipopolysaccharide (LPS)-induced shock.11,12 The different effects of thalidomide on TNF-␣ from human clinical trials and from sepsis studies in rodents may be caused by many variables. Some of these variables may involve the inflammatory condition treated. In the rodent studies factors included, the route of administration [intraperitoneal vs gavage], dose of thalidomide [6 –300 mg/kg], and the timing in which thalidomide was administrated in relation to the induction of shock [either before or after challenge with endotoxin]. Consequently, interpretation of the literature regarding the ability of thalidomide to consistently downregulate TNF-␣ in inflammatory conditions is confusing. Since the detection of cytokines such as TNF-␣ into the circulating blood volume in endotoxin models is reproducible regardless of species studied (human, primate, pig, rat, or mouse),13 we investigated the effect of
thalidomide on endotoxin-invoked synthesis of TNF-␣, interleukin (IL)-6, and IL-8 in healthy male volunteers. METHODS Subjects. Both clinical protocols were approved by an independent investigational review board. In addition, for the study involving thalidomide, the protocol was approved by the Division of Anti-infective Drug Products of the FDA and by the Hansen’s Disease Programs Research Committee. All participants in the clinical trials gave written informed consent before undertaking any study-related procedures. All subjects participating in the thalidomide trial in February 2003 were counseled on the reproduction risks associated with thalidomide and viewed a patient education video on the risks of fetal exposure associated with Thalomid® provided by Celgene Corporation (Summit, NJ). All subjects had agreed not to father a child and to wear a condom for any sexual intercourse for 1 month afterward. The originally planned, double-blinded, placebo-controlled clinical trial was permanently interrupted by severe weather (Hurricane Katrina), so it was decided to pool placebo data from a separate double-blind study performed 2 years later under identical conditions. See the Statistical Methodology section for the method used to add these placebo data. The added 18 placebo subjects did not view the video, but they did receive the 4.0-ng/kg dose of LPS that was prepared from the same lot of LPS used in the thalidomide-treated study. Endotoxin. Purified LPS (Clinical Center Reference Endotoxin, lot 2) 10,000 endotoxin units [1000 ng] per vial, obtained from the Pharmacy Development Service, Clinical Center, National Institutes of Health, Bethesda, Md, was diluted in sterile pyrogen-free water to 200 ng/mL. The LPS was administered as a bolus and at a dose of 4.0 ng/kg of body weight [the maximum dose allowed for human volunteers]. Thalomid® (thalidomide). One hundred-mg capsules and identical matching placebo capsules were kindly provided by Celgene Corporation. Thirty minutes after the last dose (100 mg p.o. q 6 h ⫻ 5 doses), the volunteers were infused with endotoxin. Study design. The protocol was a double-blind, placebocontrolled, single-site study. All subjects were confined to the research facility, beginning on the evening before study initiation and concluding 24 h after receiving endotoxin. The study was conducted in full conformance with the principles of the “Declaration of Helsinki” and its amendments, or with the laws and regulations of the locality in which the research was conducted, whichever afforded the greater protection to the individual. Assessments. Blood was collected in tubes containing sodium heparin. Before infusion of endotoxin, the plasma was collected before ingestion of thalidomide and 30 min after the last dose (0 h). After infusion of LPS, plasma was collected at 0.5, 1, 1.5, 2.5, 4,5, 6,5, 8.5, 12.5, 16.5, and 24 h and on the 7th and 30th day. For the assessment of thalidomide in the blood, the plasma was acidified using 25 mmol/L (pH 1.5) citrate buffer.14 The concentration of thalidomide was determined by high-pressure liquid chromatography using a modification of the method of Eriksson et al.15 Before and after
Translational Research Volume 150, Number 5
the infusion of endotoxin, plasma for cytokine analysis was collected at 0.0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 16, and 24 h and was frozen and analyzed by enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s procedure (Pierce Endogen Rockford, Ill). The concentrations of the cytokines were determined using software KC4V 3.3 (BioTek, Instruments, Inc., Highland Park Winooski, Vt). The areas under the curve (AUCs) were calculated using GraphPad Prism v 4.0 (San Diego Calif). Assessment of lymphocyte subsets and IL-2 and interferon-gamma (IFN-␥). Before and shortly after receiv-
ing thalidomide or placebo, blood was collected in tubes containing EDTA. The cells expressing CD3⫹/CD4⫹; CD3⫹/CD8⫹ and CD3⫹/CD69⫹ molecules was determined by using BD-conjugated MoAbs and flow cytometry analysis on a BD FACScan instrument (San Jose, Calif). Flow cytometry was performed using directly conjugated MoAbs in double-labeling and triple-labeling experiments. Appropriate fluorochromes directly-conjugated to mouse immunoglobulins were used as isotope-matched negative controls. IL-2 and IFN-␥ were assayed by ELISA kits from Pierce Endogen. Statistical methodology. The SAS statistical package (version 9.1.3; The SAS Institute, Inc., Cary, NC) was used to analyze the loge-transformed data in a repeated measures design as a split-plot arrangement of treatments with TREATMENT and SUBJECT (TREATMENT) as main plot effects. TIME and TREATMENT*TIME interactions were included on the subplot. When overall significance (P ⱕ ⫺0.05) was detected, post hoc pair-wise comparisons were performed with the Tukey HSD test for main effect comparisons and t tests of least-squares means for comparisons of interaction levels. Distributional statistics (Shapiro-Wilk test of normality, stem-leaf diagrams, box-whisker plots, and examination of individual extreme values) performed on the placebo group at each time point justified the inclusion of the additional cohort of 18 subjects into the analysis for a final placebo group of 19 subjects.
RESULTS
Thalidomide was well tolerated, and no adverse events occurred before administration of endotoxin. After the last dose of thalidomide, the mean plasma concentration of thalidomide was 1.6 g/mL. The concentrations ranged from 1.0 to 2.5 g/mL. Thalidomide remained in 3 of 4 volunteers 16.5 h after infusion with LPS at a mean concentration of (0.4 ⫾ 0.32 g/mL). After 24 h, 2 subjects had thalidomide detected at 0.34 and 0.35 g/mL. No thalidomide was detected at day 7 or day 30. The demographic characteristics of the volunteers infused with LPS are summarized in Table I. LPS evoked the synthesis of the cytokines in all volunteers (Fig 1, A–C). TNF-␣ peaked at 1.5 h after the infusion of endotoxin. This was 1 h before the peak responses for IL-8 (2.5 h) and 1.5 h before the peak response for IL-6 (3.0 h).
Shannon et al
277
Table I. The demographic characteristics of the volunteers infused with LPS A. Volunteers infused with 4 ng/kg LPS (n ⫽ 23) 1. Signed Informed Consent 2. 18–45 years of age, 65–80 kg 3. Nonsmokers, no recent history of any conditions that could affect an LPS response 4. Did not donate blood within the previous 2 months. 5. Did not receive LPS or participated in an investigational trial within the previous 30 days 6. Had a normal stable body temperature: average of 3 consecutive oral temperatures between 97.0oF and 98.8oF [Not to differ by ⬎0.4oF before challenge with LPS] B. Volunteers treated with thalidomide and infused with 4 ng/ kg LPS (n ⫽ 4) 1. Age (y) 21–43 2. Height (cm) 165–191 3. Weight (kg) 63.8–93.6
The cytokine response to LPS in the volunteers is summarized in Table II. Compared with the placebo-treated group, thalidomide did not significantly delay the appearance of the peak response of the cytokines (Fig 1, A–C). In comparing the area under the dose response curve from the 0 h to 24 h (AUC0-24) for the placebo-treated group to the thalidomide-treated group, thalidomide reduced the AUC0-24in the following order: IL-6 ⬎ TNF-␣ ⬎ IL-8 (Table II). In agreement with a previous study, IL-6 showed considerable individual variability, a peak response 2 h after a bolus injection of LPS, and a decrease that returned to baseline after 8 h.16 To reduce the variance within the groups, we used the natural log (to the base e) to transform the data. Comparing the placebo group (n ⫽ 19) to the thalidomide-treated group (n ⫽ 4), significant suppression of IL-6 was observed at 4 h (P ⫽ 0.0043), 6 h (P ⫽ 0.0145), 12 h (P ⫽ 0.0014), and 16 h (P ⫽ 0.012) (Fig 1, B). No statistically significant suppression of TNF-␣ was detected (Fig 1). It is interesting that there seemed to be a shift in IL-8. After the peak response IL-8 decreased in the thalidomidetreated group, but at 12 h, there was a shift in activity. From the 12-h to 24-h sampling of the plasma, there seemed to be an increase in IL-8 in the thalidomidetreated group. DISCUSSION
The primary objective of this study was to determine whether thalidomide could attenuate the TNF-␣ response evoked by endotoxin. Thalidomide was given before challenge with endotoxin and at doses recommended to treat acute ENL.17 Thalidomide was well tolerated, and no adverse events occurred before administration of endotoxin.
278
Shannon et al
Fig 1. Inflammatory molecules. Mean (⫾ SE) of loge -transformed data of the pg/mL of TNF-␣ (1a), IL-6 (1b), and IL-8 (1c) detected in plasma after infusion of 4.0 ng/kg of endotoxin (t ⫽ 0 h). Administration of endotoxin was preceded by ingestion of thalidomide (100 mg every 6 h ⫻ 5 doses; n ⫽ 4) [—] or 0 mg in the placebo n ⫽ 19 [- - -].
Using fluorescence microscopy, we reported that thalidomide decreased the absolute numbers of CD4⫹ cells in healthy males18 and in patients with ENL.19 In this study using flow cytometry analysis, thalidomide
Translational Research November 2007
did not significantly change the number of white blood cell/L, % lymphocytes, % granulocytes, and % monocytes, nor did it significantly alter the percent or absolute number of CD3⫹/CD4⫹; CD3⫹/CD8⫹ and CD3⫹/CD69⫹ lymphocytes (paired t test, data not shown). Thalidomide in vitro can increase mitogenstimulated mononuclear cell synthesis of IL-2 in healthy males20 and HIV-infected males.21 In this study, before and after the ingestion of thalidomide and before infusion of LPS, plasma levels of IL-2 and IFN-␥ were below the pg/mL detection levels of the standard curve on the ELISA plate (paired t test, data not shown). LPS evoked the synthesis of the cytokines in all volunteers. The appearance of TNF-␣ preceded the appearance of IL-6 and IL-8. This is in accordance with similar studies using this model.22 In our study, the cytokines at their peak responses that were most significantly suppressed by thalidomide were IL-6 and IL-8. IL-8 is a chemokine and a potent chemotaxin for neutrophils, which is the predominant cell type found in ENL skin lesions. It is produced by endothelial cells to facilitate leukocyte emigration. Dunzenforfer et al23 showed that upon stimulation with TNF-␣ or endotoxin, human umbilical vein endothelial cells (HUVECs) produce IL-8. Thalidomide, at concentrations similar to that in the plasma of the volunteers in our study, seems to have affected this process in a bidirectional manner. That is, the synthesis of IL-8 by HUVEC cells was augmented when they were stimulated with TNF-␣ and inhibited when the cells were stimulated with endotoxin. This bidirectional effect by thalidomide in this in vitro model may help explain the suppressive/enhancing effect of thalidomide on IL-8 in our in vivo study. In the endotoxin challenge model, endotoxin is cleared from the blood within 30 min after intravenous administration,24 and the major source of TNF is reported to be the liver.25 Therefore it is possible that, early in the response, endotoxin inhibited endothelial cell synthesis of IL-8, whereas later in the response TNF-␣ facilitated the synthesis of IL-8 by endothelial cells. An ability of thalidomide to regulate IL-8 secreted by endothelial cells could offer a partial explanation of its effectiveness in ENL. Recently, thalidomide has been granted FDA approval for treatment, in combination with dexamethasone, of newly diagnosed cases of multiple myeloma. The approval is based on an accelerated response rate compared with dexamethasone alone. Corticosteroids and estrogens are potent inhibitors of IL-6 production as a result of their inhibition of IL-6 gene transcription.26 It is interesting to speculate that Thalomid® (thalidomide) may synergize with dexamethasone and
Translational Research Volume 150, Number 5
Shannon et al
279
Table II. Detection of TNF-␣, IL-6, and IL-8 in the plasma of 19 placebo-treated and 4 thalidomide-treated volunteers after infusion of 4.0 ng/kg of endotoxin Cmax in pg/mL (tmax)
TNF-␣ IL-6 IL-8
AUC pg.hr/mL
% Suppression
Placebo
Thalidomide
Placebo
Thalidomide
AUC0-24
2048 (1.5 h) 2263 (3.0 h) 2338 (2.5 h)
1184 (1.5 h) 1089 (3.0 h) 1484 (2.5 h)
3370 7701 8901
2314 3378 6231
32 56 30
Abbreviations: AUC0-24, Area under the curve [(pg)(hr(0-24)/mL)] values calculated by trapezodial method; Cmax, Maximum concentration (pg/mL) for TNF-␣, IL-6, and IL – 8; % Suppression, [1 – (Mean AUC0-24 for thalidomide/(Mean AUC0-24 for placebo)] ⫻ 100; Tmax, Time in hours (0-24) to achieve maximum concentration.
markedly suppress the synthesis and/or release of IL-6. In a similar endotoxin challenge model using human volunteers, IL-6, IL-8, and TNF-␣ were significantly suppressed, in a dose response manner, by prednisolone.27 IL-6 has multiple activities, including promotion of B-cell growth and secretion of immunoglobulins by B cells, and stimulating the growth of plamacytomas, myelomas, and several B lymphomas.28 Among leprosy patients, we have shown that thalidomide can decrease IgM antibody synthesis and have speculated that this may offer an explanation for its ability to suppress an antibody-mediated Arthus-like ENL reaction.29 It seems that ENL is a hypersensitivity reaction to M. leprae having humoral and cell- mediated immune components. The roles played by antibody, complement, and sensitized T cells in precipitating ENL have yet to be elucidated. The pathogenesis of ENL is likely to be the outcome of a complex interaction of immunologic and nonspecific anti-inflammatory events. Evidence accumulating is that TNF-␣ is not the sole cytokine targeted by thalidomide in ENL and other inflammatory conditions. Recently, IL-6 but not TNF-␣ was shown to be decreased in patients treated for ENL with thalidomide.3 With the following considerations: (1) that the originally planned clinical trail was permanently interrupted by severe weather, (2) that most of the placebo data are from a separate study performed under identical conditions, and (3) that infusion of LPS mimics the ENL inflammatory response; this study adds to the evidence that downregulation of IL-6 may be a site of action of thalidomide in ENL. REFERENCES
1. Zwingerberger K, Wnendt S. Immunomodulation by thalidomide: systematic review of the literature of unpublished observations. J Inflamm 1996;46:177–211. 2. Sampaio E, Kaplan G, Miranda A, Nery JAC, Miguel CP, Viana, S, et al. The influence of thalidomide on clinical leprosum. J Infect Dis 1993;168:408 –14.
3. Villahermosa LG, Fajardo T, Abalos, RM, Balagona MV, Tan EV, Cellona RV, et al. A randomized, double-blind, doubledummy, controlled dose comparison of thalidomide for treatment of erythema nodosum leprosum. Am J Trop Med Hyg 2005;72: 518 –26. 4. Shannon EJ, Sandoval FG. Thalidomide can be either agonistic or antagonistic to LPS evoked synthesis of TNF-alpha by mononuclear cells. Immunopharmacol Immunotoxicol 1996;18:59 –72. 5. Haslett P, Roche P, Butlin C, Macdonald M, Shrestha N, Manandhar R, et al. Effective treatment of erythema nodosum leprosum with thalidomide is associated with immune stimulation. J Infect Dis 2005;192:2045–53. 6. Jacobson J, Greenspan J, Spritzler J, Ketter N, Fahey J, Jackson J, et al. Thalidomide for the treatment of oral aphthous ulcers in patients with immunodeficiency virus infection. New Engl J Med 1997;336:1487–93. 7. Wolkenstein P, Latarjet J, Roujeau, J, Duguet C, Boudeau S, Vaillant, L, et al. Randomized comparison of thalidomide versus placebo in toxic epidermal necrolysis. Lancet 1998;352:1586 – 89. 8. Sampaio E, Sarno E, Gailly R, Cohn Z, Kaplan G. Thalidomide selectively inhibits tumor necrosis factor ␣ production by stimulated human monocytes. J Exp Med 1991;173:699 –703. 9. Schmidt H, Rush B, Simonian G, Murphy T, Hsieh J, Condon M. Thalidomide inhibits TNF response and increases survival following endotoxin injection in rats. J Surg Res 1996;3:142– 6. 10. Moreira A, Wang J, Sarno E, Kapan G. Thalidomide protects mice against LPS-induced shock. Braz J Med Bio Res 1997;30: 1199 –207. 11. Ishikawa M, Kanno S, Takayangi M, Takayangi Y, Sasaki K, Thalidomide promotes the release of tumor necrosis factor-␣ (TNF-␣) and lethality by lipopolysaccharide in mice. Bio Phar Bull 1998;21:638 – 40. 12. Fernandez-Martinez E, Morales-Rios MS, Perez-Alverez V, Muriel P. Immunomodulatory effects of thalidomide analogs on LPS-induced plasma and hepatic cytokines in the rat. Biochem Pharmacol 2004;68:1321–9. 13. Hack CE, Aarden LA, Thijs LG. Role of cytokines in sepsis. Adv Immunol 1997;66:101–95. ¨ , Ekberg H. Determination of 14. Eriksson T , Björkman S, Fyge A thalidomide in plasma by high-performance liquid chromatography: avoiding hydrolytic degradation. J Chromatogr 1992;582: 211– 6. ¨ , Höiglund P. Sterospe15. Eriksson T, Björkman S, Bodil R, Fyge A cific determination, chiral inversion in vitro and pharmacokinetics in humans of the enanthiomers of thalidomide. Chirality 1995;7:44 –52. 16. Endler G, Marsik C, Joukhadar C, Marculescu R, Mayr F, Mannhalter C, et al. The interleukin-6 G(-174) C promoter
280
17. 18.
19.
20.
21.
22.
Shannon et al
polymorphism does not determine plasma interleukin-6 concentrations in experimental endotoxemia in humans. Clin Chem 2004;50:195–200. Hastings RC Leprosy. 137 1993194 Churchill Livingstone Edinburgh, UK 155. Gad SM, Shannon EJ, Krotoski WA, Hastings RC. Thalidomide induces imbalances in T-lymphocyte sub-populations in the circulating blood of healthy males. Lepr Rev 1985;56:35–9. Shannon EJ, Mestawat E, Haile-Mariam HS, Tebebe YB, Tadesse G. Thalidomide’s effectiveness in erythema nodosum leprosum is associated with a decrease in CD4⫹cells in peripheral blood. Lepr Rev 1992;63:5–11. Shannon EJ, Sandoval F. Thalidomide increases the synthesis of IL-2 in cultures of human mononuclear cells stimulated with Concanavalin-A, staphylococcal entertoxin A, and purified protein derivative. Immunopharmacology 1999;31:109 –16. Shannon EJ, Asseffa A, Pankey G, Sandoval F, Lutz B. Thalidomide’s ability to augment the synthesis of IL-2 in vitro in HIV-infected patients is associated with the percentage of CD4⫹ cells in their blood. Immunopharmacology 2000;46:175–9. Suffredini AF, Reda D, Banks SM, Tropea M, Agosti LM, Miller R. Effects of recombinant dimeric TNF receptor on human inflammatory responses following intravenous endotoxin administration. J Immunol 1995;155:5038 – 45.
Translational Research November 2007
23. Dunzenforfer S, Manfred H, Weidermann C. Inducer-specific bidirectional regulation of endothelial interleukin-8 produced by thalidomide. Immunopharmacology 1999;43:59 – 64. 24. von der Moehlen MAM, Kimmings AN, Wedel NI, Mevissen ML, Jasen J, Friedmann N, et al. Inhibition of endotoxin-induced cytokine release and neutrophil activation in humans by use of recombinant bactericidal/permeability-increasing protein. J Infec Dis 1995;172:144 –51. 25. Fong YM , Marano MA, Mokdawer LL, Wei H, Calvano SE, Denney JS, et al. The acute spanchnic and peripheral tissue metabolic response to endotoxin in humans. J Clin Invest 1990;85:1896 – 1904. 26. Hasan SI, Blaney B, Turk JL. Effect of anticancer drugs on the release of interleukin-6 in vitro. Cancer Immunol Immunother 1992;34:228 –32. 27. de Kruif MD, Lemaire LC, Giebelen IA, van Zoelen MAD, Pater JM, van den Pangaart PS, et al. Prednisolone dose-dependently influences inflammation and coagulation during human endotoxemia. J Immunol 2007;178:1845–51. 28. Akira S, Taga T, Kishimoto. Interleukin-6 biology and medicine. Adv Immunol 1993;54:1–78. 29. Shannon EJ, Miranda RO, Morales MJ, Hastings RC. Inhibition of de novo antibody synthesis by thalidomide as a relevant mechanism of action in leprosy. Scand J Immunol 1981;13:17–25.