Clinical, hematologic, and immunologic effects of interleukin-10 in humans

Journal of Clinical Immunology, Vol. 16, No. 5, 1996

Clinical, Hematologic, and Immunologic Effects of Interleukin- 10 in Humans

A M Y C. FUCHS, 1 ERIC V. GRANOWITZ, 1 LELAND SHAPIRO, 1 EDOUARD VANNIER, t GERHARD LONNEMANN, 1 J O N A T H A N B. ANGEL, 1 JEFFREY S. KENNEDY, 1 ARTHUR R. RABSON, 2 ELAINE R A D W A N S K I , 3 MELTON B. AFFRIME, 3 DAVID L. CUTLER, 3 PAUL C. GRINT, 3 and CHARLES A. DINARELLO t'4

Accepted: May 17, 1996

We conducted a double-blind, placebo-controlled study to investigate the safety, pharmacokinetics, and immunological properties of intefleukin-10 (IL-10) administration in healthy humans. Volunteers received a single intravenous bolus injection of recombinant human IL-10 (1, 10, or 25 /xg/kg) or placebo. Cytokine production in whole blood and peripheral blood mononuclear cells (PBMC) was assessed before and 3, 6, 24, and 48 hr after the injection. Peak serum concentrations of IL-10 (15 -+ 1.1,208 _+ 20.1, and 505 -+ 22.3 ng/ml) occurred after 2-5 min for 1, 10, and 25/xg/kg IL-10, respectively. The terminal-phase half-life was 3.18 hr. A transient leukocytosis (24-63% above baseline) was observed 6 hr after injection, which coincided with a dose-dependent decrease (12-24%) in neutrophil superoxide generation. There was a marked inhibition (60-95%) of endotoxin-induced IL-6 production from whole blood in each group receiving IL-10. Production of IL-8 in endotoxin-stimulated blood was reduced in the 10 /xg/kg group. In PBMC stimulated with phytohemagglutinin and phorbol ester, there was a decrease (72-87%) in interferon-,/ (IFNT) production 6 hr after IL-10 with a return to pre-IL-10 levels after 24 hr. This reduction was only partially associated with a decrease in the number of CD2-bearing cells. We conclude that IL-10 administration into humans is without significant side effects, and a single injection reduces ex vivo production of IL-6, IL-8, and 1FN'y.

KEY WORDS: Cytokines; interleukin-6; intefleukin-8; interferon-y; Thl and Th2 lymphocytes.

INTRODUCTION

Interleukin-10 (IL-10) is an important T-helper (Th) cell regulatory cytokine produced by CD4 + Th0, Th l , and Th2 and CD8 + cells (1). IL-10 suppresses cel l-mediated immunity through its inhibitory effects on T-helper type 1 lymphocytes and decreased interleukin-2 (IL-2) and interferon-3, (IFNy) production (2, 3). Suppression of Th l responses may be beneficial in several conditions such as autoimmune disease, transplant rejection, and graft-versus-host disease. In vitro IL-10 also inhibits the production of interleukin-1 (IL-1), tumor necrosis factor (TNF), interleukin-6 (IL-6), and interleukin-8 (IL-8), cytokines involved in acute and chronic inflammatory processes (2, 4 - 6 ) . Therefore, this cytokine may be useful in the treatment of acute or chronic diseases such as sepsis, arthritis, and inflammatory bowel disease.

We describe the results of a placebo-controlled, double-blind study of IL-10 administered intravenously to healthy human subjects. The safety and phannacokinet- ics after escalating doses of a single bolus injection of IL-10 were examined. We also studied the effect of IL-10 administration on neutrophil superoxide generation ex

vivo. In addition, IL-6, IL-8, IFN3~, and granulocyte macrophage-colony stimulating factor (GM-CSF) production from endotoxin-st imulated whole blood or peripheral blood mononuclear cells was measured following IL-10.

1Department of Medicine, Tufts University School of Medicine and the New England Medical Center, Boston, Massachusetts.

2Department of Pathology, Tufts University School of Medicine and the New England Medical Center, Boston, Massachusetts.

3Schering-Plough Research Institute, Kenilworth, New Jersey. 4To whom correspondence should be addressed at University of Colorado Health Sciences Center, Division of Infectious Diseases, B168, 4200 East Ninth Avenue, Denver, Colorado 80262.

291

MATERIALS AND METHODS

Volunteer Selection

Twenty-two healthy, nonsmoking male volunteers between 18 and 35 years of age were enrolled after giving

0271-9142/96/0900-0291509.50/0 �9 1996 Plenum Publishing Corporation

292 FUCHS ET AL.

informed consent. The study protocol and consent documents were approved by the Human Investigations Review Committee of the New England Medical Center and Tufts University School of Medicine. Eligibility requirements were the absence of underlying disease and a normal physical examination, chest radiograph, elec- trocardiogram, complete blood count (hemoglobin, total and differential white blood cell count, and platelets as determined using the Sysmex Toa Apparatus, Baxter Healthcare, Duarte, CA), blood chemistries (sodium, potassium, chloride, bicarbonate, glucose, blood urea nitrogen, creatinine, uric acid, calcium, phosphate, total protein, albumin, alkaline phosphatase, total bilirubin, alanine aminotransferase, aspartate aminotransferase, 7-glutamyltranspeptidase, lactate dehydrogenase, cho- lesterol, and triglycerides), and urinalysis (macroscopic and microscopic). In addition, negative viral serologies for human immunodeficiency virus type 1, hepatitis B, and hepatitis C were required. Volunteers abstained from using oral cyclooxygenase inhibitors and/or antihista- mines during the 2 weeks prior to the study and were excluded if they had taken pentoxifylline within the 3 months before the study. A negative urinalysis for the presence of illicit or prescription drugs was required for entry into the study.

Study Design

This was a randomized, double-blind, rising dose, placebo-controlled, parallel group study. Laboratory per- sonnel were unaware of the source of samples during the various analyses. Initially 18 subjects were enrolled. One volunteer in the placebo group was subsequently eliminated due to poor venous access and inability to collect blood specimens. After an interim safety analysis, four additional subjects were enrolled during a different season. The initial 17 volunteers were admitted to the Clinical Study Unit at the New England Medical Center for 60 hr, whereas the additional four volunteers were inpatients for the first 24 hr. After a 10-hr overnight fast, volunteers received either recombinant human IL-10 (Schering-Plough Research Institute, Kenilworth, NJ) or placebo (same formulation buffer without drug) as an intravenous bolus injection given over 30 sec into an antecubital fossa vein. Venous blood samples before and during the initial 6 hr following drug administration were obtained from an indwelling intravenous catheter placed in the contralateral arm. Subsequent samples were obtained by direct venipunctnre. The doses of IL-10 for the initial 17 volunteers were 1 /xg/kg (four subjects), 10 /xg/kg (four subjects), and 25 ~g/kg body weight (four subjects). Five volunteers received placebo. In the sec-

ond group of four volunteers, three received 10 /xg/kg IL-10 and one received placebo in a blinded fashion.

Clinical and Laboratoly Evaluation

Vital signs (temperature, pulse, blood pressure, and respiratory rate) were recorded prior to and 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 hr after drug administration. Volunteers were questioned during the study period for potential IL-10-related side effects. In the initial t7 volunteers, complete blood counts were obtained immediately before and 3, 6, 24, 48, and 96 hr after the injection. Blood chemistries and urinalyses were evaluated at admission and 3, 24, and 48 hr after drug administration. Blood samples for Coombs test, reticu- locyte count, and serum haptoglobin were collected before and 24, 48, and 96 hr after the injection. Plasma samples were analyzed for fibrinogen, C3, C4, and total hemolytic complement before IL-10 and 3 and 12 hr after injection. Serum IgG, IgA, and IgM were quanti- tated prior to and 96 hr after dosing. An electrocardio- gram was performed upon admission and repeated 2 and 48 hr posttreatment.

In the second group of four volunteers, blood was collected for complete blood counts before and 3, 6, 24, and 48 hr after the injection. Blood chemistries and urinalyses were evaluated at admission and 48 hr after drug administration.

Serum IL-IO Concentrations

For the pharmacokinetic study, blood was collected in sterile, vacuum tubes (Beckton-Dickinson, Rutherford, NJ) immediately prior to and 2, 5, 10, 20, 30, and 45 rain after the injection of IL-10. Additional blood samples were obtained after 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 16, and 24 hr. Blood was allowed to clot at room temperature for 20 rain and then was centrifuged for 15 rain at 4~ Serum was separated and stored at -70~ until assayed for IL-10 concentrations using an ELISA with a limit of quantitation of 100 pg/ml (Schering-Plough Research Institute).

Antibodies to IL-IO

Serum samples were obtained 12 hr before and 21 days after the injection of IL-10 and stored at -70~ Subsequently, these samples were analyzed for the presence of antibodies to IL-10 by ELISA. Briefly, wells of microtiter plates were precoated with human IL-10 and then blocked with bovine serum albumin. Serial dilutions of volunteers' sera were added. After overnight incuba-


INTERLEUKIN-10 IN HUMANS 293

tion, the wells were washed and the presence of anti- IL-10 antibodies was revealed by the addition of biotin- labeled protein A followed by strepavidin conjugated to horseradish peroxidase. The results of each sample in this ELISA were compared with the results using a pool of human serum from several normal donors. Pre-IL-10 samples were considered positive if the ratio of optical density of a volunteer's serum to optical density of the pooled serum was equal to or greater than 1.0. A post-IL-10 sample was considered positive for the presence of anti-IL-10 if the ratio of optical density of a volunteer's serum to optical density of the serum prior to IL-10 was equal to or greater than 2.0.

Isolation of Peripheral Blood Mononuclear Cells (PBMC)

Venous blood was drawn into heparinized syringes (10 U/ml) immediately before and 3, 6, 24, and 48 hr after injection of IL-10 or placebo. PBMC were isolated by centrifugation through Ficoll (Sigma Chemical Co., St. Louis, MO) and Hypaque (90%, Winthrop Laborato- ries, New York). Cells were washed twice in pyrogen- free saline (Abbott, North Chicago, IL) and resuspended at a concentration of 5 • 106 cells/ml in RPMI culture medium 1640 (Sigma) supplemented with 10 mM L- glutamine, 24 mM NaHCO 3 (Mallinckrodt, Paris, KY), 10 mM HEPES (Sigma), 100 U/ml penicillin, and 100 /~g/ml streptomycin (GIBCO Laboratories, Grand Island, NY). The complete RPMI medium was ultrafiltered through polyarnide hollow fibers to remove exogenous cytokine-inducing substances (7). PBMC were supplemented with 2% (v/v) heat-inactivated (56~ 45 min) human AB serum.

Induction of IFNT and GM-CSF Production in PBMC

Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma and diluted in DMSO at 1 mg]ml. Phytohemagglutinin (PHA) was purchased from Difco Laboratories (Detroit, MI) and diluted in RPMI. PBMC (750/xl) were aliquoted into 12 • 75-mm polypropylene tubes (Falcon, Lincoln Park, NJ) and 750 /,1 of either RPMI or PHA plus PMA diluted in RPMI (final concentration: PHA, 10/,g/ml; PMA, 100 ng/ml) were added. PBMC were then incubated for 24 hr at 37~ in a humidified atmosphere containing 5% CO2. The cultures were frozen at -70~ Prior to assay, cells were subjected to three freeze-thaw cycles for total cytokine recovery.

Fluorescent-Activated Cell-Sorting Analysis (FACS) of PBMC

PBMC were analyzed using a FACsan (Becton- Dickinson) to determine the percentage of monocytes in each sample. Monocytes were identified by size and forward scatter. To validate this method, PBMC preparations from the second group of four volunteers were analyzed using both the FACscan and the standardized Sysmex Toa Apparatus. PBMC were taken from blood collected prior to and 3, 6, 24, and 48 hr following IL-10 or placebo administration. There was no significant difference between the two methods in determining the percentage of monocytes in the P B M C (r 2 = 0.823, n = 19).

Induction of IL-8 Production in PBMC

PBMC (750 /,1) were aliquoted into 12 • 75-ram round-bottom polypropylene tubes and 750/zl of either RPMI or endotoxin (E. coli 055:B5; Sigma) in RPMI (final concentration endotoxin, 1 ng/ml) were added. PBMC were then incubated for 24 hr at 37~ in a humidified atmosphere containing 5% CO a. The cultures were frozen at -70~ Prior to assay, cells were subjected to three freeze-thaw cycles for total cytokine recovery. Since monocyte numbers increased following IL-10 administration (8), IL-8 production in the PBMC preparation is expressed as nanograms per 106 m o n o -

cytes based on the percentage of monocytes determined by the FACscan.

Isolation of Neutrophils

After density-gradient centrifugation through Ficoll and Hypaque, the interphase cells and overlying plasma were removed leaving the neutrophil-red cell pellet. The neutrophil-red blood cell pellet was suspended in 2 vol of pyrogen-free saline containing 3% dextran (200,000 MW; Sigma) and sedimented by gravity. The supernatant was removed, centrifuged, and washed in saline. The remaining red blood cells in the neutrophil pellet were subjected to hypotonic lysis in water (4~ for 20 sec and then reconstituted to 0.9% using 9% NaC1. The neutrophils were washed and resuspended in cold Hanks' balanced salt solution (HBSS) without phenol red at a concentration of 107 cells/ml.

Superoxide Release from Neutrophils

Superoxide was measured by the reduction of ferricy- tochrome C to ferrocytochrome C (9). Neutrophils (final


294 FUCHS ET AL.

concentration 5 X 106/ml) from the initial 17 subjects were aliquoted into 1.5-ml Eppendorf tubes with 1.2 mg/ml cytochrome c (Sigma) and incubated for 15 min in a 37~ water bath under one of the following conditions: (A) with 100 ~g/ml superoxide dismutase (SOD; Sigma) in HBSS, (B) with 500 ng/ml PMA in HBSS, (C) with 500 ng/ml PMA in HBSS plus SOD (100 /~g/ml), and (D) with HBSS alone. Following the incubation, tubes were centrifuged for 1 rain at 10,000g to pellet the cells. The supematant was transferred to a 96-well microplate. Cytochrome c reduction was determined by measuring the optical density at 550 nm. Induction of superoxide was calculated by subtracting the difference between the value of SOD from both PMA-stimulated and control incubations.

Whole Blood Cytokine Production

One milliliter of either RPMI or endotoxin diluted in RPMI was added to polypropylene tubes prior to the addition of 2.5 ml of heparinized blood (final concentration endotoxin, 10 ng/ml). Blood was incubated upright for 24 hr at 37~ in 5% CO 2. After incubation, the supernatant plasma was transferred into sterile 1.5-ml microfuge tubes and centrifuged for 2 min at 10,000g at room temperature. The resulting platelet-poor plasma was removed and frozen at -70~ in potypropylene tubes.

Cytokine Assays

Samples were analyzed for IL-1/3 (10), TNFa (11), IL-6 (12), IL-8 (13), TNF-soluble receptor p55 (TNF- sRp55) (14), and IL-1 receptor antagonist (IL-1Ra) (15) by specific radioimmunoassays. The limit of quantifica- tion of the radioimmunoassays for each cytokine were as follows: IL-1/3, 40 pg/ml; TNFa, 80 pg/ml; IL-6, 40 pg/ml; IL-8, 80 pg/ml; TNFsRp55, 160 pg/ml; and IL-1Ra, 80 pg/ml. IFN7 levels in PBMC were determined by ELISA (R&D Systems, Minneapolis, MN) with a limit of detection of 62 pg/ml. GM-CSF levels in PBMC were determined by ELISA (Genzyme Inc., Cambridge, MA). The limit of detection for GM-CSF was 10 pg/ml.

Statistical Analysis

IL-10 serum levels for the three dose groups are expressed as the mean _+ SE. Cytokine data obtained at 3, 6, 24, and 48 hr for each volunteer were expressed as the percentage of the baseline (t = 0) value (set at 100%) for each subject. Percentages are shown as the mean +_

SE. Statistical analyses were based on a comparison of data at times 3, 6, 24, and 48 hr with baseline values. Statistical analyses were performed by analysis of vari- ance (ANOVA) using Fisher's least significance difference or by Student's t test.

RESULTS

Clinical and Laboratory Evaluation

The schedule of clinical and laboratory evaluations for the initial 17 volunteers is depicted in Table I. There were no adverse symptoms reported by volunteers injected with IL-10. In addition, there were no significant differences in blood chemistries, urinalyses, hemoglobin concentrations, platelet counts, or coagulation parameters between any dose-level group receiving IL-10 and the placebo-injected volunteers.

One volunteer with a previous history of detergent allergy developed a faint, erythematous, maculopapular rash on his back and arms 3 days after administration of 10 /zg/kg of IL-10. Five days after injection, the rash resolved. The rash was present exclusively in those skin areas that had contact with the bed sheets. A second volunteer developed mild first-degree atrioventricular (AV) block (PR interval, 203 msec) 2 hr after administration of 10 /~g/kg IL-10. His PR interval returned to baseline 48 hr after injection (PR, 189 msec). A third volunteer had mild first-degree AV block (PR interval, 220 msec) prior to injection. Two hours after administration of 25 /zg/kg IL-10, his PR interval increased to 233 msec. However, 48 hr after injection his PR interval had decreased to normal (190 msec).

Table II shows the changes in complement compo- nents, fibrinogen, and immunoglobulin levels for the initial 17 volunteers. There was a significant increase (P < 0.01) in C4 levels in both the placebo and the 10 /xg/kg IL-10 groups 12 hr after injection. Since these elevations were not seen following the 1 or 25 ~g/kg doses, their clinical significance is unclear. There were no differences in the other complement component, fibrinogen, or immunoglobulin levels following injection of IL- 10 or placebo.

P harmac okinetic s

The IL-10 serum concentration-time curves are shown in Fig. 1. Serum IL-10 concentrations were not measured in the second group of four volunteers. Serum IL-10 concentrations in the placebo-injected subjects were less than 0.1 ng/ml at all time points. One subject in the 1

Journal o f Clinical Immunology, Vol. 16, No. 5, 1996

Tab

le I

. S

ched

ule

of

Stu

dies

in

Vol

unte

ers

Rec

eivi

ng I

L-1

0

Tim

e (a

fter

bol

us i

njec

tion

of

IL-1

0)

Eva

luat

ion

-12

hr

0 2

min

5

min

1

0m

in

20

rain

3

0m

in

45

min

lh

r 1

.5h

r 2

hr

2.5

hr

3h

r 4

hr

5h

r 6

hr

8h

r 1

2h

r 1

6h

r 2

4h

r 3

6h

r 4

8h

r 9

6h

r 21

day

s

>

�9

" C

linic

al

eval

uati

on

Sym

ptom

s an

d vi

tal

sign

s x

x In

ject

ion

site

ev

alu

ati

on

x

Ele

ctro

card

iogr

am

x C

linic

al

labo

rato

ry

CB

C,

diff

, pl

atel

ets

x x

Blo

od

chem

istr

ies

x U

rina

lysi

s x

C3,

C4,

CH

50

x S

erum

IgG

, Ig

M,

IgA

x

Coo

mbs

tes

t x

Ret

icul

ocyt

e c

otm

t X

Hap

togl

obin

x

Fib

rino

gen

x S

erum

IL

-IO

le

vels

x

IL-1

0 an

tibo

dy

x Im

mun

olog

ic

test

s P

BM

C a

nd

who

le b

lood

cy

toki

nes

x N

eutr

ophi

l su

pero

xide

ge

nera

tion

x

X X

X X

X X

X X

X X

X

X

X

X

X

X

X

X

X

X

X

X

X X

X X

X X

X X

X X

X X

X X

X X

X X

X X

X

x X

x X

X

X

X

X

X

X

Volunteer Laboratory test group 0hr 3 hr 12hr 96 hr

Table II, Laboratory Data in Volunteers Receiving IL-10 or Placebo

Mean concentration • SE following injection of IL-10 or placebo

Significance

a Not significant. b Compared to time 0.

C3 (mg/dl) Placebo 87.6 • 6.2 1/xg/kg 87.5 -+ 2.7

10/*g/kg 90.3 + 5.8 25/*g/kg 94.8 -+ 4.8

C4 (mg/dl) Placebo 15.6 • 1.7 1/xg/kg 17.0 -+ 1.3

10/xg/kg 17.0 -+ 2.3 25/xg/kg 17.0 -+ 0.9

CH50 (U) Placebo 136.6 -+ 11.7 1/xg/kg 110.8 -+ 16.1

lO/xg/kg 127.5 • 13.9 25/zg/kg 159.0 -+ 9.3

Fibrinogen (mg/dl) Placebo 238.8 -+ 14.7 1/*g/kg 235.0 -+ 10.7

10/xg/kg 227.0 • 21.1 25/xg/kg 247.3 -+ 14.8

IgG (mg/dl) Placebo 969.0 -+ 82.7 1/xg/kg 1008.0 -+ 111.9

10/xg/kg 860.3 -+ 70.7 25/xg/kg 1187.3 -+ 96.2

IgM (mg/dl) Placebo 107.6 -+ 10.7 1/xg/kg 116.3 -+ 17.9

10/xg/kg 114.3 _+ 26.6 25/zg/kg 76.0 _+ 11.6

IgA (mg/dl) Placebo 259.2 -+ 30.0 1/xg/kg 210.3 -+ 21.7

10/xg/kg 190.5 -+ 20.5 25/*g/kg 277.3 -+ 74.2

86.2 -+ 5.0 91.6 -+ 5.3 84.2 _+ 2.3 86.0 _+ 2.2 92.5 _+ 4.3 94.3 _+ 6.4 99.8 _+ 4.8 95.5 +- 2.9 15.6 _+ 1.7 18.0 _+ 2.0 16.3 _+ !.1 18.8 _+ 2.3 17.5 _+ 2.3 19.8 -+ 2.3 18.5 -+ 1.0 18.8 • 1.1

133.8 -+ 11.0 142.8 _+ 15.2 105.3 • 11.8 96.0 -+ 20.3 148.3 _+ 8.2 156.3 _+ 9.4 168.5 _+ 12.8 164 _+ t0.4 243.4 + 15.5 229.6 _+ 14.1 226.3 _+ 13.8 222.0 _+ 8.6 226.5 _+ 17.5 212.0 _+ 34.3 263.8 -+ 13.6 271.3 _+ 29.0

951.6 • 90.1 1005.7 _+ 152.3 846.5 +_ 56.3

1154.5 _+ 74.0 102.4 -+ 11.7 112.0 _+ 17.9 105.3 -+ 23.6 73.0 _+ 8.4

256.0 _+ 29.2 211.0 _+ 31.0 183.3 • I3.7 283.3 _+ 79.5

NS a NS NS NS

P < 0.01 b NS

P < 0.01 b NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS

1000

/zg/kg group was eliminated from the pharmacokinetic analysis due to the presence of interfering factors in his serum. Mean concentrations of IL-10 at 2 min (C a rain)

100

k " ! gg/kg �9 10 gg/kg a 25 btg/kg

10

o 1

.1

296 FUCHS ET AL.

4 8 1 '2 1 '6 2'0 2'4

H o u r s

Fig. 1. Pharmacokinetics of IL-10 in healthy humans. Volunteers were injected with 1, 10, or 25/zg/kg IL-10 as a single bolus injection at time 0. Serum IL-10 levels were measured by ELISA. Data are depicted as mean -+ SE. The dashed line represents the limit of quantitation of the IL-10 ELISA.

were 13.9 • 2, 156 • 31, and 491 • 19 ng/ml for the 1, 10, mad 25 /xg/kg groups, respectively. Peak serum concentrations (Cma• were reached 2-5 min following injection. The mean peak concentrations of IL-10 after bolus injection were 15 __ 1.1 ng/ml for the 1/xg/kg dose group, 208 -+ 20.1 ng/ml for the 10/xg/kg dose group, and 505 • 22.3 ng/ml for the 25 /xg/kg dose group. Although IL-10 was quantifiable at all doses studied, rigorous pharmacokinetic analysis was not possible in the 1 and 10 /zg/kg groups due to the absence of a definitive elimination phase. Therefore, half-life, volume of distribution, and clearance were calculated based on mean data from the 25 /xg/kg group. The half-life was 3.18 hr and IL-10 distributed into a volume of 0.26 L/kg following bolus injection. The clearance of IL-10 was 0.93 ml/kg/min.

Antibodies to IL-IO

Antibodies to recombinant IL-10 were not detected in the sera of any volunteers either before the IL-10 injection or after 21 days.



g

175[ - A ~ Placebo

/ '* 1 gg/kg 150]

1

50 . . . . . 0 12 2 4 3'6 4'8

�9

175

150'

125

10@

75'

50' 0 12 2 4 36 4 8

175-

150-

..~ �9

125 -

g 100~

75-

50-

C ~ A 25 ~tg/kg

12 24 36 48 Hours

Fig. 2. Effect of IL-10 on the peripheral WBC. Absolute numbers of

white blood cells were calculated immediate ly before and 3, 6, 24, and

48 hr after injection of (A) placebo or 1/xg/kg, (B) 10/xg/kg, or (C) 25

/xg/kg IL-10. For each volunteer, WBC after IL-10 administration are

expressed as the percentage (mean _+ SE) of cells compared with t ime 0 (100%). Differences were analyzed for significance by ANOVA:

*P < 0.05; **P < 0.01; ***P < 0.001.

Effect of lL-lO on the Peripheral WBC

Significant elevations in the absolute WBC were observed 6 hr following injection of IL-10 during both study periods. As shown in Fig. 2, the increase above baseline was 24% in the 1 ~g/kg group (P < 0.01; Fig. 2A), 63% in the 10 ~g/kg group (P < 0.001; Fig. 2B), and 47% in the 25/xg/kg group (P < 0.01; Fig. 2C). Of

note, there was also a 12% rise at 6 hr in the placebo- injected group (P = 0.044). As reported elsewhere, this leukocytosis was due to an absolute neutrophilia and monocytosis, not a lymphocytosis (8).

Superoxide Release from Neutrophils

Unlike the placebo-injected subjects, neutrophil superoxide generation at 6 hr was depressed (12-24%) in a dose-dependent fashion in volunteers receiving IL-10 (Fig. 3). Interestingly, this 6-hr time coincides with the leukocytosis. The decrease, however, did not reach statistical significance (P = 0.096, P = 0.052, and P = 0.105 for the 1, 10, and 25 /xg/kg groups, respectively). The 3- and 24-hr time points did not reveal any change in superoxide release from the baseline level.

Effect of lL-lO on Whole Blood IL-6 and IL-8 Production

Whole blood cytokine production was measured in all subjects at the time points shown in Table 1. There was no change in the production of IL-1/3, TNF-c~, IL-6, IL-8, TNFsRp55, or IL-1Ra from baseline (predrug) levels during a 24-hr incubation in the absence of endotoxin (data not shown).

In contrast, marked inhibition of endotoxin-induced IL-6 production occurred in subjects given IL-10. In the low-dose group (1 ~g/kg), 60% inhibition of baseline production was observed 3 hr after the injection (P < 0.01); production returned to preinjection levels by 6 hr (Fig. 4A). In the 10 ~g/kg group, there was a 92% reduction in endotoxin-induced IL-6 production at 3 hr (P < 0.001; Fig. 4B). In this group, inhibition of IL-6 persisted 6 hr after IL-10 administration (76%; P < 0.001). In addition, 15-20% inhibition was present 48 hr after the injection (P < 0.06 and P < 0.02 at 24 and 48 hr, respectively). In the high-dose group (25 /xg/kg), inhibition was nearly complete (91-95%; P < 0.001) at 3 and 6 hr and was still present 48 hr after the injection (47%; P < 0.001) (Fig. 4C). Similar suppression of TNFc~ and 1L-1/3 production has been reported following IL-10 administration (8).

There were no significant changes in IL-8 production following injection of 1 or 25 ~g/kg IL-10 (Figs. 5A and C). Statistically significant reductions in IL-8 production in whole blood were observed at 3, 24, and 48 hr in the 10 ~g/kg group (Fig. 5B). However, similar reductions were seen at 24 and 48 hr in volunteers receiving placebo (Fig. 5A).


298 FUCHS ET AL.

175 / A --------o----- Placebo

t �9 1 l.tg/kg

i 150

o ~ 1 2 5 ~ @ ~ ~ ~ ~ ~

~E

0 6 1 2 1 8 2'4

g '9,

175- A

150"

125"

100~ i 75"

50"

25"

0 0

Placebo �9 1 gg/kg

1'2 2'4 3'6 4'8

i1 ~ 1 o 1~ % 75

0 6

�9 10 gg/kg

1'2 1'8 2'4

g '9,

125]

100~

50 .

0 1 '2 2'4

�9 10 gg/kg

3'6 4'8

.~ 175] c

15o] ~, 1 2 5 ] ,

N ~

75

Z

0 6

Fig. 3.

25 ~tg/kg

1 '2 1 '8 2'4 Hours

Effect of IL-10 on neutrophil superoxide generation. Neutro- phils were isolated from volunteers immediately before and 3, 6, and 24 hr after (A) placebo or 1/xg/kg, (B) 10 txg/kg, or (C) 25/xg/kg IL-10. For each volunteer, PMA-induced superoxide generation is expressed as a percentage (mean _+ SE) compared to time 0.

Effect of lL-lO on Production of lL-8 by PBMC

IL-8 production in PBMC was measured both in the initial 17 volunteers and in the second group of 4 subjects. There was no significant change in IL-8 production in endotoxin-Stimulated peripheral blood mononuclear cells following administration of IL-10 (10 or 25 /xg/kg) or placebo (Figs. 6A-C). Analysis of IL-8 production was not performed in the 1 /_~g/kg group due to a technical error in two of the volunteers in that group.

e, g

175[ ~ C A 25 Ixg/kg /

12 t 1001

ol . . . . . . .

0 12 2,1 36 48 Hours

Fig. 4. Effect of IL-10 on IL-6 production in whole blood stimulated with endotoxin. Heparinized blood was collected from volunteers immediately before and 3, 6, 24, and 48 hr after (A) placebo or 1/xg/kg, (B) 10/xg/kg, or (C) 25/zg/kg IL-10. Blood was incubated for 24 hr in the presence of endotoxin (10 ng/ml). IL-6 levels in the supematant plasma are expressed as a percentage (mean _+ SE) of the levels at time 0. Differences were analyzed by ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001.

Effect of lL-lO on IFN3, Production in PBMC

IFN 7 production in PBMC was measured both in the initial 17 volunteers and in the second group of 4 subjects (Figs. 7A-C). IFN? was depressed by 71% at 3 hr and 87% at 6 hr in volunteers receiving 10 /xg/kg IL-10, although due to subject variability, these de-



oo

200

175

1504

125

100

75

50

25

o~

- - - - o - - - - P l a c e b o

�9 1 Ixg/kg

12 24 36

I 48

225]A

15Ol 125

100 ,d

7 5

50

25

-----o--- Placebo

1 '2 2'4 3 '6 4'8

%-

150]

50

25

0 I . , . 0 1 2 2'4

�9 10 I.tg/kg

I

3 '6 4'8

225

200

"~" 175 .9 "B I50

125 g I00'

75-

50-

25-

" �9 10 p , g / k g

12 24 36 48

225] 200! i

175 ~

150 i

125"

100,

75"

50"

25-

0"

C 25 gg/kg

0 12 24 36 4 8

Hours

Fig. 5. Effect of IL-10 on IL-8 production in whole blood stimulated with endotoxin. Heparinized blood was collected, stimulated, and incubated as described in the legend to Fig. 4. For each volunteer, IL-8 released after administration of (A) placebo or 1 /xg/kg, (B) 10/xg/kg, or (C) 25 ~g/kg IL-10 is expressed as a percentage (mean +_ SE) of the production at time 0. Differences were analyzed by ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001.

creases did not reach statistical significance (P = 0.095) using ANOVA. However, when comparing the data at 3 and 6 hr to time 0 using the paired t test, P values of 0.041 and 0.037 were obtained, respectively. There was also a 72% decrease in IFN 7 production at 6 hr in the group receiving 25/xg/kg IL-10 (P = 0.17 using a paired t test). Since PHA stimulates T cells via the CD2 receptor

225] 12

200"I

i ,5o11

100

7 5

0

a 25 lxg/kg

t t 1 '2 2'4 3 '6 4'8

Hours

Fig. 6. Effect of IL-10 on IL-8 production in PBMC stimulated with endotoxin. PBMC were isolated from volunteers immediately before and 3, 6, 24, and 48 hr after a bolus injection of (A) placebo or (B) 10 p,g/kg, or (C) 25/xg/kg of IL-10. Cells were stimulated with endotoxin (1 ng/ml) for 24 hr. For each volunteer, endotoxin-induced IL-8 production was calculated as nanograms per 106 monocytes as described under Materials and Methods. The data are expressed as a percentage (mean -+ SE) of the production at time 0. Differences were analyzed by ANOVA.

and CD2-bearing lymphocytes decreased after IL-10 administration (8), we calculated IFN-y levels per 106 CD2 cells. A 65% reduction in IFN3, levels persisted at 6 hr in the 10 /xg/kg group despite a 25% reduction in CD2-positive cells in the PBMC cultures compared to predrug levels.


300 FUCHS ETAL.

g

700

600'

500

4 0 0

300

200

100 X

Placebo

12 24 36 48

e~

g

300

200

100

�9 10 gg/kg

1 2 2 4 3 6 4 8

700

600

500

.~ 4oo

3oo

200

100~

0-

A 25 ge,/kg

12 24 36 Hours

48

Fig. 7. Effect of IL-10 on IFN 7 production in PBMC. PBMC were isolated from volunteers immediately before and 3, 6, 24, and 48 hr after a bolus injection of (A) placebo or (B) 10/zg/kg or (C) 25/xg/kg IL-10. Cells were stimulated with PHA (10 /xg/ml) and PMA (100 ng/ml) for 24 hr. IFNT production was calculated per 2.5 • 106 PBMC. Levels are expressed as a percentage (mean -+ SE) of the production at time 0. Differences were analyzed by paired t test: *P < 0.05.

Effect o f lL-lO on GM-CSF Production in PBMC

GM-CSF production in PBMC was measured in the initial 17 subjects before and after IL-10 or placebo. There were no significant changes in GM-CSF production by PBMC in response to stimulation by PHA (10 /zg/ml) plus PMA (100 ng/ml) (data not shown).

DISCUSSION

The addition of IL-10 to cultured human monocytes or neutrophils stimulated in vitro results in inhibition in the synthesis of IL-1, TNF, IL-6, and IL-8 (5, 6). In addition, when added to T lymphocyte cultures, IL-10 suppresses mitogen and antigen driven proliferation and inhibits the synthesis of IL-2 and IFN7 (2, 3). These data suggest that IL-10 may have a therapeutic role in several acute and chronic inflammatory diseases as well as for diseases involving cell-mediated immunity such as transplant rejection. The present study was conducted to determine whether the in vitro properties of IL-10 can be observed in the cells of healthy velunteers given intravenous IL-10. The clinical safety, immunogenicity, and effects on host parameters of homeostasis were also investi- gated.

Fifteen healthy males received a single bolus injection of IL-10 (1, 10, or 25 /xg/kg) and six subjects were injected with placebo. No clinically significant adverse effects were observed in the IL-10 group. IL-10 reached a mean Cmax between 2 and 5 min after the bolus injection. Similar times to peak concentrations have also been observed after intravenous injection of other cytokines (16). There were no significant differences in the hemoglobin concentrations, platelet counts, or blood chemistries in the groups receiving IL-10 versus placebo. Although there was a significant increase in the C4 concentration in the 10 #g/kg group 12 hr after IL-10 injection, a similar increase was seen in the placebo group. There was no increase in C4 in the 1 or 25/xg/kg groups, suggesting that this is not an effect of IL- 10. Two volunteers developed prolongation of the PR interval following IL-10 administration. The effects on PR interval appeared minimal and it is unclear whether IL-10 has an effect on AV node conduction. We conclude that administration of IL-10 as a single bolus injection appears safe.

Quantitative IgG, IgA, and IgM levels were un- changed 96 hr after IL-10 administration. In vitro,

however, IL-10 stimulates Ig production when added to B cell cultures (17). In fact, when IL-10 was added to B cell cultures from patients with common variable immunodeficiency, a disease characterized by inadequate production of immunoglobulins, increased synthesis of IgG, IgA, and IgM was observed (18). In those studies, the B cells were preactivated with anti-CD40 or Staphylococ-

cus aureus Cowan strain in vitro. In our study, B cell activators or antigens were not coadminsitered in vivo or added in vitro. The possibility exists that if IL-10 is given to patients with a deficiency in Ig production, it may



result in increased immunoglobulin production by B cells in vivo.

There were significant elevations (24-63% above baseline) in the peripheral WBC 6 hr after injection of IL-10. As reported previously, neutrophils and monocytes were significantly increased (40-160%), while lymphocyte numbers were lower (20-30%) (8). Cell numbers returned to baseline at 24 hr. It is possible that the increases in WBC may represent either an IL-10- mediated release from the bone marrow or a reduction in the expression of endothelial adhesion molecules resulting in redistribution of leukocytes. The latter hypothesis is supported by a model of autoimmune lung injury in rats in which aerosolized IL-10 decreased pulmonary vascular ICAM-1 expression and led to a decline in neutrophil transmigration into the alveoli (19). Leukocy- tosis associated with stress hormones is rapid (within 60 rain) and is an unlikely explanation of this phenomenon.

There was a dose-dependent trend toward decreased neutrophil superoxide generation 6 hr after IL-10 administration; however, this did not reach statistical significance. The finding suggests that IL-10 may have a direct effect on neutrophil superoxide release or the decreased superoxide generation may reflect a different population of neutrophils. The latter explanation is supported by the increased number of circulating neutrophils 6 hr after IL-10 injection. These neutrophils may be an immature population released from the bone marrow or an older population which demarginated during IL-10 treatment. One explanation is that immature neutrophils have a reduced ability to generate superoxide. Another explanation is that there may be "leukocyte exhaustion" in older, marginating neutrophils.

After 24 hr of incubation in RPMI, we found no spontaneous IL-1, TNF, IL-6, IL-8, or IL-1Ra production in whole blood drawn 3, 6, 24, or 48 hr after IL-10 injection. These data confirm in vitro studies that IL-10 does not induce the synthesis of cytokines in monocytes, T lymphocytes, or neutrophils.

In each group receiving IL-10, endotoxin-induced synthesis of IL-6 in whole blood was significantly inhibited. Sixty to ninety-five percent inhibition was observed 3 and 6 hr after injection of IL-10. As reported previously, a similar inhibition of both IL-1 and TNF was observed in these same IL-10-injected volunteers (8). Some of the inhibition of IL-6 production may be a consequence of residual IL-10 present in the whole blood samples. It appears, however, that most of the inhibition cannot be explained by residual IL 10. For example, in the 10 kcg/kg group, 69% inhibition of IL-6 production occurred at 6 hr when the mean serum concentration of IL-10 was 0.8 ng/ml. We have observed that the presence

of 8 ng/ml or more of IL-10 in vitro is required to suppress 50% of endotoxin-induced IL-6 production in whole blood (unpublished observation). Furthermore, despite undetectable levels of IL-10 in serum 24 hr after the injection in the high-dose group, IL-6 synthesis was still suppressed by 47% at 48 hr. These findings suggest that IL-10-induced cytokine suppression may persist for at least 48 hr.

IL-6 is an endogenous pyrogen (20), is a potent stimulator of B cell growth and immunoglobulin production (21), and plays an important role in the acute phase response (22). IL-6 has been implicated in the pathogenesis of B cell malignancies and autoimmune diseases. Specifically, IL-6 has been shown to play a role in the pathogenesis of multiple myeloma, plasmacytomas, rheumatoid arthritis, atrial myxoma, and Castelman's disease (23-28). Anti-IL-6 antibodies have been used as therapy for some of these conditions (28). Since IL-10 inhibits IL-6 production, treating these conditions with IL-10 should be considered.

In contrast to IL-6, production of IL-8 in both whole blood and PBMC was only minimally affected by IL-10 administration. In vitro, IL-10 inhibits both PBMC and neutrophil production of IL-8 (5, 6). In volunteers receiving IL-10, absolute monocyte numbers increased 6 hr following injection. Therefore, we calculated IL-8 production in PBMC cultures per 10 6 monocytes. How- ever, if the monocytosis is due to bone marrow release of new cells, less mature cells may produce more IL-8 in response to endotoxin. In fact, small, immature monocytes produce more IL-1 than mature cells (29). Thus, a reduction in IL-8 production per monocyte may have been masked by an increase in IL-8 synthesis per new cell.

Decreased IFNy production was observed in stimulated PBMC 6 hr after in vivo administration of 10 or 25 /xg/kg IL-10. Part of the reduction may be explained by a decrease (25-30%) in the fraction of lymphocytes comprising the PBMC preparations 6 hr after injection of IL-10 compared to the fraction of lymphocytes before the drug (8). In particular, lymphocytes bearing the CD2 surface marker were reduced (12-25%) at 6 hr. Although PHA triggers T cell signaling through the CD2 receptor, only a small portion of the reduction in IFNy production can be accounted for by the decreased number of CD2 lymphocytes in the PBMC cultures. For example, in the 10 /xg/kg group, an 87% reduction in IFNy production occurred at 6 hr. When IFNT synthesis was calculated per 106 CD2 cells, a 65% decrease in production persisted. Similarly, in the 25 ~g/kg group, a 72% decrease in IFNy production was observed at 6 hr and a 66% reduction persisted when calculated per 10 6 CD2 cells.


302 FUCHS ET AL.

Thus, IL-10 inhibits ex vivo IFN~/production by both reducing the number of lymphocytes in the PBMC cultures and, more significantly, by directly inhibiting its synthesis.

GM-CSF production in stimulated PBMC was unaf- fected by in vivo administration of IL-10. This is in contrast to reports that IL-10 inhibits GM-CSF production when added to PBMC cultures stimulated with PHA or anti-CD3 (2). However, our present study differs from these in vitro experiments since PMA was used in addition to PHA as a stimulus.

In this study we have shown that a single injection of IL-10 is safe in humans. In addition, the pharmacokinetics following bolus injection are defined. We have also demonstrated that IL-10 induces a moderate leukocytosis and is a potent inhibitor of ex vivo IL-6 production.

ACKNOWLEDGMENTS

T h e au thors t h a n k A u b r e y E. Boyd , Pa t r i c ia M. Noga ,

D o n n a Keany , T i m o t h y C u m m i n g s , and the staff o f the

Cl in ica l S tudy U n i t o f the N e w E n g l a n d Med i ca l Center .

W e are a lso gra tefu l for the a s s i s t ance of Scot t F.

Orenco le , G i a m i l a Fantuzz i , X i - X i a n Z h a n g , H e i - D e

W e n , E l l en C. Dona ldson , and Ph i l ip A. K o n n i k o v . W e

w o u l d also l ike to t h a n k Dr. She i la J acobs and R o n Sabo

of S c h e r i n g P l o u g h R e s e a r c h Ins t i tu te and the staff o f the

Cl in ica l I m m u n o l o g y L a b o r a t o r y o f the N e w E n g l a n d

M e d i c a l C e n t e r for the i r i nva luab le ass is tance . Th i s w o r k

was suppor ted by Na t iona l Ins t i tu tes of Hea l th Gran t s

AI -15614 , AI -07329 , M O 1 R R 0 0 0 5 4 - 0 3 3 , and R O 1 - A I -

33290 (E .V.G. ) and b y the S c h e r i n g - P l o u g h R e s e a r c h

Inst i tute . G e r h a r d L o n n e m a n n is suppor t ed b y D F G L o

535/1-1 .2 .

REFERENCES

1. Yssel H, De Waal Malefyt R, Roncarolo M, Abrams JS, Lahesmaa R, Spits H, De Vries JE: IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells. J Immunol 149:2378-2384, 1992

2. Viera P, De Waal Malefyt R, Dang MN, Johnson KE, Kastelein R, Fiorentino DF, De Vries JE, Roncarlo MG, Mosmann TR, Moore KW: Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: Homology to Epstein-Barr virus open reading frame BCRFI. Proc Natl Acad Sci USA 88:1172-1176, 1991

3. Taga K, Tosato G: IL-10 inhibits T cell proliferation and IL-2 production. J Immunol 148:1143-1148, 1992

4. Dinarello CA: Biologic basis for interleukin-1 in disease. Blood 87:2095-2147, 1996

5. de Waal Malefyt R, Abrams J, Bennett B, Figdor CG, de Vries JE: Interleukin 10 (IL-10) inhibits cytokine synthesis by human

monocytes: An autoregulatory role of IL-10 produced by monocytes. J Exp Med 174:1209-1220, 1991

6. Cassatella MA, Meda L, Bonora S, Ceska M, Constantin G: Interleukin 10 (IL-10) inhibits the release of proinflammatory cytokines from human polymorphonuclear leukocytes. Evidence for an autocrine role of tumor necrosis factor and IL-1/3 in mediating the production of IL-8 triggered by lipopolysacchaxide. J Exp Med 178:2207-2211, 1993

7. Schindler R, Dinarello CA: Ultrafiltration to remove endotoxins and other cytokine-inducing materials from tissue culture media and parenteral fluids. Biotechinques 8:408-413, 1990

8. ChemoffAE, Granowitz EV, Shapiro L, Vannier E, Lonnemann G, Angel JB, Kennedy JS, Rabson AR, Wolff SM, Dinarello CA: A randomized, controlled trial of interleukin-10 in humans: Inhibi- tion of inflammatory cytokines and immune responses. J Immunol 154:5492-5499, 1995

9. Klempner MS, Dinarello CA, Henderson WR, Gallin JI: Stimula- tion of neutrophil oxygen-dependent metabolism by human leukocyte pyrogen. J Clin Invest 64:996-1002, 1979

10. Cannon J, Van der Meer J, DK, Endres S, Lonnemann G, Burke J, Dinarello C: Interleukin-1/3 in human plasma: Optimization of blood collection, plasma extraction, and radioimmunoassay methods. Lymphokine Res 7:457-467, 1988

11. van der Meer JWM, Endres S, Lonnemann G, Cannon JG, Ikejima T, Okusawa S, Gelfand JA, Dinarello CA: Concentrations of immunoreacfive human tumor necrosis factor-~ produced by human mononuclear cells in vitro. J Leukoc Biol 43:216-223, 1988

12. Schindler R, Mancilla J, Endres S, Ghorbani R, Clark SC, Dinarello CA: Correlations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood 75:40-47, 1990

13. Porat R, Poutsiaka DD, Miller LC, Granowitz EV, Dinarello CA: Interleukin-1 (IL-1) receptor blockade reduces endotoxin and Borrelia burgdo~feri-stimulated IL-8 synthesis in human mononuclear cells. FASEB J 6:2482-2486, 1992

14. Shapiro L, Clark BD, Orencole SF, Poutsiaka DD, Granowitz EV, Dinarello CA: Detection of tumor necrosis factor soluble receptor p55 in blood samples from healthy and endotoxemic humans. J Infect Dis 167:1344-1350, 1993

15. Poutsiaka DD, Clark BD, Vannier E, Dinarello CA: Production of interleukin-1 receptor antagonist and interleukin-1/3 by peripheral blood mononuclear Cells is differentially regulated. Blood 78: 1275-1281, 1991

16. Bocci V: Interleukins: Clinical pharmacokinetics and practical implications. Clin Pharmacokinet 21:274-284, 1991

17. Rousset F, Garcia E, Defrance T, Peronne C, Vezzio N, Hsu D, Kastelein R, Moore KW, Bancherean J: Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes. Proc Natl Acad Sci USA 89:1890-1893, 1992

18. Zielen S, Bauscher P, Hofmann D, Meuer SC: Interleukin 10 and immune restoration in common variable immunodeficiency [let- ter]. Lancet 342:750-751, 1993

19. Mulligan MS, Jones ML, Vaporciyan AA, Howard MC, Ward PA: Protective effects of IL-4 and IL-10 against immune complex- induced lung injury. J Immnnol 151:5666-5674, 1993

20. Helle M, Brakenhoff JP, De GER and Aarden LA: Interleukin 6 is involved in interleukin 1-induced activities. Eur J Immunol 18: 957-959, 1988

21. Hirano T, Taga T, Nakano N, Yasukawa K, Kashiwamura S, Shimizu K, Nakajima K, Pyun KH, Kishimoto T: Purification to



homogeneity and characterization of human B cell differentiation factor (BCDF or BSFp-2). Proc Natl Acad Sci USA 82:5490- 5494, 1985

22. Gauldie J, Richards C, Harnish D, Lansdorp P, Banmann H: Interferon/32/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells. Proc Natl Acad Sci USA 84:7251-7255, 1987

23. Kawano M, Hirano T, Matsuda T, Taga T, Horii Y, Iwato K, Asaoku H, Tang B, Tanabe O, Tanaka H, Kuramot0 A, Kishimoto T: Autocrine generation and requirement of BSF-2/IL-6 for human multiple myelomas. Nature 332:83-85, i988

24. Nordan RP, Potter M: A macrophage-derived factor required by plasmacytomas for survival and proliferation in vitro. Science 233:566-569, 1986

25. Houssiau FA, Devogelaer J-P, Van Damme J, De Deuxchaisnes CN, Van Snick J: Interleukin-6 in synovial fluid and serum of patients with rheumatoid arthritis and other inflammatory arthriti- des. Arth Rheum 31:784-788, 1988

26. Hirano T, Taga T, Kiyoshi Y, Nakajima K, Nakano N, Takatsuki F, Shimizu M, Murashima A, Tsunasawa S, Sakiyama F, Kishi- moto T: Human B-cell differentiation factor defined by an anti- peptide antibody and its possible role in autoantibody production. Proc Nail Acad Sci USA 84:228-231, 1987

27. Yoshizaki K, Matsuda T, Nishimoto N, Kuritani T, Taeho L, Aozasa K, Nakahata T, Kawai H, Tagoh H, Komori T, Kishimoto S, Hirano T, Kishimoto T: Pathological significance of interleukin 6 (IL-6/BSF2) in Castelmans' disease. Blood 74:1360-1367, 1989

28. Beck JT, Hsu S, Wijdenes J, Bataille R, Klein B, Vesole D, Hayden K, Jagannath S, Barlogie B: Brief report: Alleviation of systemic manifestations of Castelman's disease by monoclonal anti-interleukin-6 antibody. N Engl J Med 330:602-605, 1994

29. Elias JA~ Schreiber AD, Gustilo K, Chien P, Rossman MD, Lammie PJ, Daniele RP: Differential interleukin 1 elaboration by unfractionated and density fractionated human alveolar macro- phages and blood monocytes: Relationship to cell maturity. J Im- munol 135:3198-3204, 1985


Documents

Clinical, hematologic, and immunologic effects of interleukin-10 in humans