into the regulation of platelet production ...

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1996 88: 3354-3362

Differential mechanisms in the regulation of endogenous levels of thrombopoietin and interleukin-11 during thrombocytopenia: insight into the regulation of platelet production M Chang, Y Suen, G Meng, JS Buzby, J Bussel, V Shen, C van de Ven and MS Cairo

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Differential Mechanisms in the Regulation of Endogenous Levels of Thrombopoietin and Interleukin-11 During Thrombocytopenia: Insight Into the Regulation of Platelet Production By Mei Chang, Yu Suen, Gloria Meng, Jeffrey S. Buzby, Jim Bussel, Violet Shen, Carmella van de Ven, and Mitchell S. Cairo The regulation of megakaryocytopoiesis and thrombopoiesis appears t o be under the control of an array of hematopoietic growth factors. To determine the relationship of endogenous thrombopoietic cytokine levels and circulating platelet (PLT) counts, we measured the levels of thrombopoietin (TPO), interleukin-11 (IL-111, and interfeukin-6 (IL-6) in patients with significant thrombocytopenia secondary to both marrow hypoplasia and increased PLT destruction. Increased endogenous levels of TPO and IL-11, but not IL-6, were detected in bone marrow transplant patients with thrombocytopenia following myeloablative therapy (BMT/ MAT) (TPO: 1,455.5 f 87.3 pg/mL, [PLT 39,600 f 7,8OO/pLl, P c .001, n = 12; IL-11: 227.9 f 35 pg/mL, [PLT 32,900 k 5,7OOO/pL], P c .05, n = 19; IL-6 25.8 f 8.4 pg/mL, [PLT 32,800 f 5,O57/pL], P > .05, n = 4) Y normal donors (TPO c150 pg/mL, n = 8; IL-11 .05, n = 62). In patients

with immune thrombocytopenia purpura (ITPI, with decreased PLT survival, but intact bone marrow megakaryocytopoiesis, endogenous IL-11 levels were significantly in3,OOO/pLl, P creased (328.0 k 92.6 pg/mL. [PLT 20,900 < .05, n = 25). However, endogenous TPO levels remained undetectable ( ~ 1 5 0 pg/mL, [PLT 30,500 f 5,50O/pL], n = 15). These results suggest that there may be differential mechanismsregulating endogenousTPO, IL-11, and IL-6 levels during acute thrombocytopenia and suggest that the absolute number of circulating PLTs may not always be the sole regulator of endogenousTPO levels. Other mpl-expressing cells of the megakaryocyte lineage may contribute t o the regulation of circulating TPO levels as well. Our results also suggest IL-11 levels may in part, be regulated by a negative feedback loop based on circulating PLT counts, but also may, in part, be regulated by a variety of inflammatory agonists. Both TPO and IL-11, therefore, appear t o be active thrombopoietic cytokines regulating, in part, megakaryocytopoiesis during states of acute thrombocytopenia. 0 7996 by The American Society of Hematology.

T

receptor, the product of the c-Mpl proto-oncogene.”“ Expression of c-Mpl in mice and humans appears to be restricted to PLTs megakaryocytes, and late progenitors of megakaryocytic lineage.9~’2~’’ TPO binding induces tyrosine phosphorylation of a number of substrates, such as Jak2, Shc, and the Mpl receptor itself in human platelet and megakaryocytic cell line^.'^-'^ In addition to its importance in signal transduction, both in vivo and in vitro data suggests that Mpl receptor may be involved in the uptake and metabolism of TPO by PLTS.’~.’’ A family of pleiotropic hematopoietic growth factors, with a common signal transduction pathway including interleukin- I I (IL- 1 I), IL-6, and leukemia inhibitory factor, has been shown to have various stimulatory effects on megakaryocytopoiesis and PLT production.”,*’ Within this family, IL1 1 represents a unique polypeptide, initially cloned from a primate marrow stromal cell line (PU-34).” Preclinical studies in both rodents and nonhuman primates have .demonstrated that IL-I 1 induces significant enhancement of megakaryocytop~iesis.~~-~~ Administration of IL-11 also results in a significant elevation in the PLT count of neonatal rats? bone marrow transplanted mice,z6 and splenectomized mice.24Recently, a randomized placebo-controlled trial of recombinant human IL-11 (50 Fgkg) in adult cancer patients with a history of PLT transfusions during severe thrombocytopenia secondary to chemotherapy demonstrated a significant reduction in patients requiring PLT transfusions during subsequent cycles of ~hemotherapy.2~ Several lineage-dominant humoral factors that regulate the homeostasis of specific peripheral blood cells have been shown to have an inverse correlation between their circulating level or activity and the respective/responsive blood cell mass, 17.28-34 We have previously demonstrated a significant inverse correlation of circulating granulocyte colony-stimu-

HE LIGAND for the c-Mpl proto-oncogene, which is predominantly produced in the liver and kidney, has recently been purified, cloned from several species, and expressed in mammalian cells.’-6 Recombinant Mpl ligand (thrombopoietin) (PO)has been shown to both enhance megakaryocyte development and to increase the size, number, and ploidy of developing megakary~ctes.~-~~’~* Intraperitoneal injection of mice or neonatal rats with purified ligand results in a 400% increase in the circulating platelet (PLT) c o ~ n t . ~ .TPO, ~ , ’ like many other hematopoietic growth factors, exerts its biologic effects through binding of a specific

From the Division of Hematology/Oncology and Blood and Marrow Transplantation, Children’s Hospital of Orange County, Orange, CA; Genentech, S. San Francisco, CA; and New York Hospital, Cornell University, New York, NY. Submitted March 12, 1996; accepted June 24, 1996. Supported by grants from the Pediatric Cancer Research Foundation (Orange, CA), the Walden W. and Jean Young Shaw Foundation (Chicago, IL), and the Children’s Hospital of Orange County Research and Education Foundation (Orange, CA). Presented in part at the Society of Pediatric Research San Diego. CA, May 7-11, 1995 and at the American Society of Hematology, Seattle, WA, December 1-5, 1995. M.C. and Y.S. contributed equally to this manuscript. Address reprint requests to Mitchell S. Cairo, MD, Director, Hematology/Oncology Research and Blood and Marrow Transplantation, Children’s Hospital of Orange County, 455 S. Main St, Orange, CA 92668. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. 0006-4971/96/8809-~0009$3.00/0 3354

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Blood, Vol 88,No 9 (November l ) , 1996: pp 3354-3362

From bloodjournal.hematologylibrary.org by guest on July 21, 2011. For personal use only. TPO AND 11-11 LEVELS DURING THROMBOCYTOPENIA

3355

Table 1. Demoaraohics of BMT Patients Patient

No.

1 2 3 4

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Age (yr)/Sex

12lM 121M 111M 71M 14lF 1OIM 31M 8/M 20lM 1OIF 2lF 13lM 21M 25/F 11F 3lF 31M 4lF 91F 3lF 71M 6lF 6lM 91F

Diagnosis/ Disease Status

ALU2CR ALU2CR ALU2CR NB/R,D, Ewing's/R,D, FBSC/R,D, PNET/R,D, ALU2CR ALU3CR ANLU3CR ANLUlCR ANLUlCR GEM/R,D, A.SJICR ESG/R,D, AAJICR ANLUlCR NHU2CR ESG/R,D, ANLUlCR ALU3CR ANLUlCR AAJICR NB/R,D,

Conditioning Regimen*

BMT

Cytokine Administration?

GVHD Prophylaxis+

TBI. VP-16/VPL, CY TBI, VP-1WPL. CY TBI, VP-16NPL. CY TBI, VP-16/VPL, CY Mel, CY; CPL, VP-16 Mel, CY, CPL, VP-16 CPL, TT; VP-16 TBI, VP-16 TEI, VP-16, ARA-C T, ARA-C, EU VP-16, ARA-C, CY, TT TEI, VP-16, CY ECNU, TTNP-16 ECNU, TTNP-16 ECNU, TTNP-16 ATG, CY BUICY ECNU, CYNP-16 TTNP-16 BUICY TEI, VP-16, CY TBI, VP-16, CY ATG, CY TEI,VP-16/VPL, CY

Auto Auto Auto Auto Auto/PSCT AutoIPSCT AutoIPSCT All AlloIPSCT AlloIMRD AlloIMUD AlloIM RD AutolPSCT AutoIPSCT AutolPSCT AllolM UD AlloIMRD AlloIMRD Auto AlloIMRD AlloIMRD AlloIMRD AllolMRD Auto

GMIG GMIG GMIG GM G G GM GM GM GM GM GMIG GM GM GM GM NA NA NA NA GM GM NA GM

NA NA NA NA NA NA NA CSPIMTX T-cell Depl MTX T-cell Depl CSPIMTX NA NA NA CPSIMTX MTX CPSIMTX NA MTX MTX MTWCPS CPSIMTX NA

Abbreviations: AA, aplastic anemia; ALL, acute lymphoblastic leukemia; ALLOIMRD, allogeneic EMTImatched related donor; ALLOIMUD, allogeneic BMTImatched unrelated donor; ANLL, acute nonlymphoblastic leukemia; ARA-C, cytosine arabinoside; AS, anaplastic astrocytoma; AUTO, autologous BMT; AUTOIPSCT, autologous EMTlperipheral stem cell transplant; ESG, brain stemglioma; BU, busulfan; CPL, carboplatin; CR. complete remission; CSP. cyclosporin; Cy, cyclophosphamide; Ewing's, Ewing's sarcoma; FBSC, fibrosarcoma; NB, neuroblastoma; NHL, non-Hodgkin's lymphoma; G, granulocyte; GEM, glioblastoma; GM, granulocyte-macrophage; Mel, melphalan; MTX, methotrexate; PNET, primitive neuroectodermal tumor; R,D, residual disease; T-cell Depl, T-cell depletion; TBI, total body irradiation; TT, thiotepa; VP, etoposide; VPL, verapamil; NA, not applicable. Conditioning Regimens: TBI 1,200 cGy, BID x 3d; VP 1,800 m g h 2 d; VPL ,005 mg/kg/min; CY 60 mg/kg/d x 2d. TEI 1,200 cGy, BID x 3d; VP 1,800 mglm'ld; CY 60 mglkgld x 2d. TEI 1,200 cGy, BID x 3d; VP 750 mglm'ld x 2d; ARA-C 3 glm' EID x 3d; CY 45 mg/kg/d x 2 d. TEI 1,200 cGy, BID x 3d; VP 1.800 mglm'ld. MEL 50 mglm' x 4d; CY 250 mglm' x 4d; CPL 300 mglm'ld; VP 2,000 mglm'ld CI x 4d. CPL 500 mglm2/d x 3d; TT 10 mglkgld x 3d; VP 8.3 mglkgld x 3. VP 750 mglm'ld x 2d; ARA-C 3glm' BID x 3d; CY 1,350 mglm'ld x 2d; TT 250 mglm'ld x 3d. TT 250 mglm'ld x 3d; busulfan 4 mgikgld x 3d; ARA-C 3 glm' BID x 2d. BCNU 100 mglm', BID x 3d; TT 300 mg/m'/d, x 3d; VP 250 mglm2/d, x 3d. CY 60 mglkgld, X 2d: ATG 20 mglkgld, x 2d. EU 4 mglkgld. x 4d; CY 60 mgikgld. x 2d. CY 60 mgikgld, x 2d; VP 260 mglm'ld, x 3d; BCNU 100 mglm', BID x 3d. VP 250 mglm'ld, x 3d; TT 300 mglm'ld, x 3d. t Cytokines: G-CSF 10 pglkgld. GM-CSF 250 pglm'ld followed by G-CSF10 pglkgld (WBC ==3OOlpL).GM-CSF 250 pglm2/d. GVHD Prophylaxis: MTX 10 mglm'ld, days 1,3,6,11. CSP 3 mglkgld; MTX 10 mglm'ld, days 1,3,6, 11. T-cell depletion: €-Rosette + soybean agglutination.

*

lating factor (G-CSF) with neutropenia35in patients undergoing bone marrow transplant (BMT) after myeloablative therapy (MAT). To determine the relationship of endogenous thrombopoietic cytokine levels with circulating PLT counts and the mechanisms associated with the regulation of these cytokine levels, we measured the circulating levels of TPO, IL-11, and IL-6 in patients with significant thrombocytopenia secondary to both marrow hypoplasia and increased PLT destruction. MATERIALS AND METHODS MAT/BMTpatient plasma samples. Between January 1992 and May 1995, a total of 481 plasma samples from 24 patients, all of whom received MAT and BMT as part of the treatment for their malignancies, were collected for subsequent analysis. The characteristics of their disease status and treatment are listed in Table 1. Plasma samples from MATBMT patients were obtained from each

patient before administration of myeloablative therapy; on the day of BMT; every other day thereafter until absolute neutrophil count was ==5OO/pL;every week thereafter until PLT count was ~50,0001 pL; and every month thereafter until 12 months post-BMT. Submyeloblative therapy. Between April 1994 and July 1994, plasma samples from seven pediatric cancer patients who received submyeloablative therapy (SMAT) were collected for subsequent analysis. The patients, four women and three men, ranged from 2 to 27 years of age (median age, 8 years). Three patients had acute lymphoblastic leukemia (ALL) and the other four patients had either acute nonlymphoblastic leukemia (ANLL), astrocytoma, Wilm's tumor, or rhabdomyosarcoma. Plasma samples from SMAT patients were obtained during acute thrombocytopenia episodes (PLT < 50,OOO/pL). Immune thrombocytopenia purpura patient plasma samples. Between May 1991 and November 1994, a total of 55 plasma samples from 29 patients with immune thrombocytopenia purpura (ITP) were collected for subsequent analysis. The clinical and laboratory data

From bloodjournal.hematologylibrary.org by guest on July 21, 2011. For personal use only. 3356

CHANG ET AL Table 2. Demographics of ITP Patients

Patient

No.

Age (yrMSex

PLT (k/wL)

Treatment

21F 51M 31F 1OIF 111M 111F 111F

12 8 20 32 43 18 7 47 51 46 13 4 25 3 6 10 11 10 11 6 8 11 28 21 34 22 5 19 16 18.86 t 2.6

IVIGISteroids IVlG IVlG

_____

1* 2* 3' 4* 5*

6* 7*

8* 9t

lot llt 12t 13t 14* 15* 16t 17t 18t 19t 20' 21" 22* 23* 24* 25'

26* 27*

28" 29*

Mean t SD

31M 61M 111F 13lF 7lM 31M 2lF 3lM 3lF 41M 11M 12lF 8lM 41M 7lM 17lF 71M 11F 51F 91F 16/M 111F 7.1 t 0.8

WIG

WIG WIG IVlG IVlG IVIGISteroids IVIGISteroids IVlG IVlG IVlG IVlG WIG IVlG IVlG IVlG NIA WIG WIG WIG NIA NIA NIA NIA NIA IVlG IVlG NIA

Abbreviations: NIA, not available; IVIG, intravenous gamma globulin; Steroids, prednisonelprednisolone. * Newly diagnosed. t Recurrent.

for those patients are listed in Table 2. Plasma samples from newlydiagnosed ITP patients were obtained at diagnosis, 3 to 21 days after diagnosis while PLT counts recovered (2100,00O/pL),and 6 months after diagnosis. Plasma samples from recurrent ITP patients were obtained during thrombocytopenia episodes, as well as after PLT recovery. Consent, clinical data, and plasma sample collection. Eight ITP patients were treated at New York Hospital, Comell Medical Center, New York, NY, between May 1991 and September 1994. All other patients were treated at Children's Hospital of Orange County, Orange, CA. Informed consents for blood samples were obtained from the patients or parentdguardians as approved by the Institutional Review Board of both hospitals. Clinical and laboratory data of the patients were obtained from the patient's medical records. Plasma was separated, aliquoted, and stored frozen at -80°C immediately after blood samples were collected, until assay. Enzyme-linked immunosorbent assay (ELISA) for TPO. The TPO levels in plasma were measured by a TPO receptor-antibody mediated sandwich ELISA. A full description of the assay will be published elsewhere.36Briefly, microtiter plates were coated at 4°C overnight with 100 pL of rabbit F(ab') antihuman Fc (2 mgInL, Jackson Immunoresearch, West Grove, PA) and then incubated for 2 hours at room temperature with 100 pL of a chimeric molecule consisting of human TPO receptor fused to the Fc portion of human lg G (100 ng/mL, Genentech, S. San Francisco, CA). Twofold serial dilution (initial dilution 1:s) of plasma samples and standards were

added to the wells and incubated for 1 hour. Bound TPO was quantitated colorimetrically by incubation with 100 pL of biotinylated affinity-purified polyclonal rabbit antihuman TPO (Genentech) followed by streptavidin-conjugated peroxidase (Vector, Burlingame, CA) and substrate. Full-length, glycosylated, recombinant human TPO produced by mammalian cells was used to generate standard curves, which were subjected to four parameter nonlinear regression curve fitting. The sensitivity range of the assay was 150 to 1,000 pglmL of TPO. The assay does not cross react with human LL-6 and IL-I 1 . The performance characteristics of the ELISA were similar in plasma and serum and independent of the type of anticoagulant used. The ELISA preferentially detects full-length TP0.37TPO levels measured by ELISA were further validated by comparable results using the megakaryoblastic HU-3 cell proliferation assay.'x There is a significant positive correlation ( P < ,001) between the stimulatory activity of thrombocytopenic sera determined by HU-3 bioassay and TPO levels determined by the ELISA." Mixing rhTPO at a concentration of 1,000 pg/mL with representative plasma samples and measuring the recovery of rhTPO did not show any interfering substances in the plasma. ELISA for detecting human IL-11. Levels of IL-I I were measured by a sandwich ELISA.39Briefly, microtiter plates were coated with monoclonal anti-rhIL-I1 antibody (Genetics Institute, Cambridge, MA). RhIL-11 standards, blanks, and test samples were added and incubated at room temperature. Biotinylated mouse antiIL- I1 monoclonal antibody (Genetics Institute) was added and incubated at room temperature. Plates were washed and avidin-conjugated horseradish peroxidase (Vector, Burlingame, CA) was added and incubated for 1 hour at room temperature. Plates were washed and o-phenylenediamine (Sigma, St Louis, MO) was added as the substrate. The reaction was stopped after S minutes by addition of 2.25 m o l n sulfuric acid. Optical density of the samples was measured at 490 nm with a Bio Rad (Richmond, CA) EIA reader. Sensitivity of the assay ranged from 40 to 1,000 pg1mL of IL-1 I . Various concentrations of rhIL-I1 (0 to 5,000 pg1mL) were used for the standard curve. All samples were run in duplicate. IL-1 I levels by ELISA were previously validated by correlating the standard curve with a B 9/11 proliferation assay (performed by Frann Bennett, Genetics Institute).'") ELISA jor detecting human IL-6. Levels of 1L-6 were measured by a sandwich ELISA (Biosource International, Camarillo, CA) according to manufacturer's instructions. Sensitivity of the assay ranged from 2 pg/mL to 500 pg/mL. All samples were run in duplicate. Statisricaf analysis. Results are expressed as mean values 2 standard error of the mean (SEM) of three or more samples. Where appropriate, the probability of significant differences between two groups was determined using the unpaired Student's t-test. The relationship between platelet and cytokine levels was analyzed by linear correlation (Spearman Correlation Test) and regression analysis. Statistical analyses were performed using the InStat statistical program (Graph Pad, San Diego, CA) for the Macintosh computer. P values < .OS were considered significant. RESULTS

T P O levels in the plasma of MAT/BMTpatients. To determine if plasma levels of TPO protein were altered during thrombocytopenia, 215 plasma samples from 12 patients undergoing MATBMT were assayed for TPO. The mean concentrations of plasma TPO from the 12 patients and the corresponding mean number of PLTs as a function of time following MAT are shown in Fig 1A. Before MAT (day -7) with normal PLT counts (211,500 -+ 26,10O/~L),plasma TPO levels were undetectable ( .371, NS) (Fig 3B). TPO levels in the plasma of SMAT. A total of nine plasma samples collected from seven patients with thrombocytopenia (median PLT counts 62,OOO/pL; range, 36,00O/pL to 118,00O/pL) secondary to SMAT were assayed for TPO. Significantly elevated TPO levels were observed in all but one sample (median TPO level, 689 pg/mL, range, 361 to 1,227 pg/mL, P < .01).

100

PLT (k/mm3)

500

Fig 2. (A) Time course of IL11 levels and PLT counts in 19 cancer patients undergoing BMT after MAT. Pre-MAT day corresponds to day -7. MAT days correspond to days -7 to -1. BMT day corresponds to day 0, posttransplantation period corresponds to days +1 to +360. Mean k SEM of the values are presented. *IL-11 levels at day -7 were all below the minimum detectable level (40pglmL) and the actual value of the data is not known. (6) Correlation between plasma IL-11 levels and PLT counts in cancer patients undergoing BMT after MAT. The results of 249 samples from 19 patients were plotted. A significant correlation between plasma IL11 levels and PLT count was found ( r = -0.329, P i .00011 using Spearman correlation analysis.

IL-1I levels in ITP patients. The plasma levels of IL- 1 I from children with acute ITP at diagnosis were significantly increased when compared to age-matched controls (IL- I 1: 328.0 -+ 92.6 pg/mL, PLT: 19,000 -t 2,7OO/pL, n = 25, P < .05) (Fig 4). Fourteen days after diagnosis, PLT counts recovered following treatment. The endogenous IL- 1 1 levels, however, remained elevated (IL-11: 530.4 2 159.7 pg/ mL, PLT: 241,000 -+ 38,2OO/pL, n = 13, P < .05), similar to the BMT patients. Six months after diagnosis, the circulating IL-1 I levels had returned to normal levels (66.4 2 20.3 pg/mL, PLT: 282,800 t 46,200/pL, n = 7). TPO levels in the plasma of ITP patients. A total of 15 plasma samples collected during thrombocytopenia (median PLT counts 30,5OO/pL; range, 6,OOO/pL to 52,0OO/pL) from 15 ITP patients were also assayed for TPO. In contrast to the results with IL- 11, all of the ITP patients at diagnosis had undetectable circulating TPO levels ( .05 (NSII using Spearman correlation analysis.

60

0

-

9 d

O O

d 100

10

30

ri T

0

0

4

r = -0.1147 p = 0.371 (NS) n=62

t.

0 I

I

I

,

I

I

I

I

10

30

50

70

90

110

130

I -

150

0

0

50

1

I

10

'

" " " ' I

100

'

" I

500

PLT (Ut")

Day

gous peripheral blood progenitor cell transplantation was measured by a semiquantitative 32D/huMpl+ bioassay system. Although the patients in that study had a shorter duration of severe thrombocytopenia (about 5 days compared with about 30 days in our patients), an inverse correlation between TPO levels and PLT count, over time, was demonstrated and is consistent with the results presented here?' Conversely, the TPO levels observed in ITP patients with severe thrombocytopenia remained undetectable (Fig 4). A mechanism was recently proposed suggesting that in patients with normal liver function, TPO production is constitutive and circulating TPO levels are controlled by the circulating

1600 11-11

1400

600

1200

500

1000

z

?

0

E?

800

-E!

0

-9

F?

sessed to evaluate the relationship of endogenous TPO levels to the circulating platelet count. Our patient population could be divided into two major groups: MAT/BMT and SMAT patients with thrombocytopenia, decreased PLT production, and bone marrow hypoplasia; and ITP patients with thrombocytopenia, decreased PLT survival, and increased PLT production based on increased bone marrow megakaryoc y t e ~ . ~Elevated ' TPO levels from patients with severe thrombocytopenia following myeloablative therapy indicated an inverse relationship between circulating TPO levels and the circulating PLT count. In a recent study, TPO bioactivity in serum from patients undergoing MAT and autolo-

700

0

-E

.E

-I

B

400

800

300

600

200

400

100

200

c

2

0

0 age-matched Ped

ITP Dx

ITP Day +14

ITP Day +180

3?

--3

Fig 4. Endogenous 11-11 levels from pediatric acute ITP patients at diagnosis (Dx) In = 25); 14 days after diagnosis (n = 13); and 180 days after diagnosis (n = 7). Mean f SEM of the values are presented. *Mean f SEM of IL-11 levels of controls versus ITP Dx, P c .05; **mean f SEM of 11-11 levels of control versus ITP day +14, P < .002. PLT control 203,000 f 7,5001pL; ITP Dx 19,000 2 2,0001pL; ITP day +14 241,000 2 38,2001pL; ITP day +180 282,800 f 46,2001 pL. I--------) Minimum detectable level of TPO. TPO levels were all below the minimum detectable level and the actual value of the data is not known.


PLT mass through Mpl receptor-mediated uptake and metabolism. Consequently, a lower circulating PLT mass would have less capacity for TPO uptake and metabolism, resulting in higher circulating TPO 1 e ~ e l s . Our l ~ results from ITP patients, together with the recent findings that Mpl receptor is expressed not only on PLTs but also on the megakaryocyte and its progenitor^,'^ however, suggest that the absolute number of circulating PLTs may not be the sole regulator of endogenous TPO levels. We propose that circulating TPO levels may be regulated by the total cell mass of the megakaryocyte lineage that expresses Mpl receptor. Therefore, circulating TPO levels may be significantly increased in thrombocytopenic patients with bone marrow hypoplasia due to low amounts of total Mpl receptor-expressing cellular mass. In contrast, despite similar degrees of acute thrombocytopenia, the total number of Mpl receptors, and consequently the TPO catabolism capacity in ITP patients, may be higher due to increased numbers of megakaryocyte progenitor cells, megakaryocytes, and the continuous production of new PLTs, which may still be able to take up TPO before autoimmune clearance, resulting in low or undetectable levels of TPO. Consistent with our hypothesis is the recent observation that low levels of circulating TPO have been demonstrated in transcription factor, NF-E2, knock-out mice, which have a maturation defect with PLT production, but retain high megakaryocyte Ievel~.~' The possible contribution of altered Mpl receptor numbers per cell andor expression patterns of different Mpl receptor variants to the undetectable circulating levels of TPO in ITP patients is currently under investigation.44Additionally, since increased bone marrow megakaryocytes were observed at diagnosis in all of our ITP patients, it is possible that circulating TPO levels had briefly surged before diagnosis. Because TPO levels could only be measured after diagnosis, this surge may have been missed. Finally, it is possible that small differences in the TPO levels in control patients compared with ITP patients may not have been observed, as our assay did not detect TPO levels below 150 pg/mL. More sensitive ELISAs are being developed to determine if such differences ~ c c ~ r However, . ~ ~ . ~such ~ , ~ brief or low level increases would still be considerably different responses than that observed in patients with hypoplastic bone marrows. Elevated IL- 11 levels were observed in both MATBMT patients and in ITP patients (Figs 2 and 4). A significant inverse correlation between circulating PLT counts and endogenous IL- 11 levels was observed in the MAT/BMT patients ( r = -0.329, P < .0001). Bellido et and others have recently demonstrated that IL- 1 1 receptors are expressed on megakaryocytic and bone marrow stromal cells.4x We and others have also reported that IL- 11 production can be induced by a number of inflammatory agonists, including IL-la, transforming growth factor (TGF) pl and TGFp2.49,50 Elevated IL-11 levels in ITP patients indicated that unlike TPO, endogenous IL-11 levels may not be regulated by IL11 receptor expressing cells. Decreased PLT count in both MAT-treated and ITP patients may be one of the regulators inducing IL- 11 production. The prolonged elevation of IL11, despite PLT recovery, may be due to other inflammatory agonists produced during thrombocytopenia. The relation-

CHANG ET AL

ship between the IL-11 elevation and the expression of these cytokines in vivo, therefore, requires further investigation. IL-6 is a multifunctional cytokine that increases in vitro megakaryocyte size and megakaryocyte ploidy when combined with IL-3 in ~ i t r o . ~ 'Preclinical .~' in vivo studies of IL-6 demonstrated increased PLT production in both mice and primate^.^^,^' Endogenous IL-6 levels in patients with thrombocytopenia secondary to MAT in our study, however, were not correlated with circulating PLT counts (Fig 3). Our data corroborates previous results that demonstrated the lack of any relationship of circulating PLT counts and IL-6 levels,ss557 In summary, both TPO and IL- 11 appear to be important physiological regulators of megakaryocytopoiesis and thrombopoiesis. Endogenous IL-11 levels appear to be regulated by both circulating PLT count and possibly other inflammatory agonists, whereas endogenous TPO levels appear to be regulated by the total Mpl receptor-expressing cellular mass. Further studies are needed to determine the mechanism associated with TPO absorption and metabolism by Mpl receptor expressing cells. Future clinical trials of both TPO and IL-I 1 will help delineate the importance and role of each of the thrombopoietic cytokines in regulating megakaryocytopoiesis, particularly in the context of ITP. ACKNOWLEDGMENT The authors thank Kit Garcia and Andrea Hebert at Genentech for their technical assistance for the TPO ELISA analysis, and Drs Dan Eaton, Frederic de Sauvage, Mary Napier, and Wai Lee Wong at Genentech and Dr Robert Emmons at National Institutes of Health for their helpful discussions in the preparation of this maunscript. We also thank Renee Dulak, Sally Anderson, and Linda Rahl for their editorial assistance in the preparation of this manuscript, and Robin Ellis and Marilyn Perez for sample and data collection.

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