Department of Pharmacology, Hunterian Institute, Royal College of Surgeons, Lincoln's Inn Fields, London. WC2A 3PN, UK. Received for publication 20 June ...
Br. J. exp. Path. (I989) 70, 93-IOI
Increased vascular permeability and polymorphonuclear leucocyte accumulation in vivo in response to recombinant cytokines and supernatant from cultures of human synovial cells treated with interleukin Ij* M.L. Watson, G.P. Lewis and J. Westwick Department of Pharmacology, Hunterian Institute, Royal College of Surgeons, Lincoln's Inn Fields, London WC2A 3PN, UK
Received for publication 20 June I988 Accepted for publication i i October I988
Summary. The inflammatory effects of intradermal injections of the human recombinant cytokines interleukin i (IL-i), granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNFcx) have been assessed in rabbit skin, and compared with the effects of a novel polymorphonuclear leucocyte (PMN)-stimulating activity (PSA) produced by IL-i-treated human synovial cell cultures. IL-i (84 fmol) and GM-CSF (io pmol) caused increases in vascular permeability with a delayed onset, as assessed by the dermal accumulation of intravenously-administered 125I-human serum albumin. These cytokines also stimulated extravascular accumulation of PMNs. In contrast, PSA-containing supernatant caused a more rapid and prolonged increase in vascular permeability and PMN accumulation. TNFa (84 fmol) was unable to stimulate either of these responses. The increases in vascular permeability and PMN accumulation following IL-i administration in vivo may be a consequence of the local generation of PMN-stimulating activity by connective tissue cells, such as the activity produced by IL-i-treated synovial cell cultures that we have described.
Keywords: cytokines, interleukin i, polymorphonuclear leucocyte, inflammation, synovial cell Interleukin i (IL-i), and other members of the family of polypeptide mediators known as cytokines, have been implicated in several aspects of the inflammatory response. In vitro IL-i stimulates cultured cells to produce inflammatory mediators such as plateletactivating factor (Bussolino et al. I 986) and prostaglandins (Albrightson et al. I985),
and increases endothelial-leucocyte adhesion (Bevilacqua et al. I985). IL-i was thought to possess intrinsic polymorphonuclear leucocyte (PMN)-stimulating activities (Klempner et al. I978, I979; Sauder et al. I984), but experiments performed with recombinant IL-I have failed to confirm these findings (Watson et al. I98 7b; Georgilis
A portion of this work has been presented to the British Pharmacological Society and has been published in abstract form (Watson M.L., Lewis G.P. & WestwickJ. (I988) Cytokine-induced increases in rabbit skin vascular permeability. Br. 1. Pharmacol 93, 144P). Correspondence: M.L. Watson, Department of Applied Pharmacology, National Heart and Lung *
Institute, Dovehouse Street, London SW3 6LY, UK.
93
M. L. Watson et al. 94 GM-CSF (4 x I07 colony-stimulating units/ et al. I987). Despite these results, human recombinant IL-i does stimulate extravascu- mg) were generous gifts from Drs Stern and lar PMN accumulation in vivo (Pettipher et al. Lomedico (Hoffman La Roche, Nutley, NJ, I986; Movat et al. I987) and in an attempt USA); Dr Miiller (Ciba-Geigy, Basel, Switzerland); and Drs Gillis and Henney (Immunex, to reconcile these findings we recently described a novel PMN-stimulating activity Seattle, WA, USA) respectively. These cyto(PSA) in the supernatant of IL-I-treated kines were diluted in sterile PBS/o. 2 5% BSA. human synovial cell cultures (Watson et al. TNFa (6 x 107 cytolytic units/mg) was a 198 7a). This activity is distinct from eicosa- generous gift from Dr Adolf (Boehringer noid products of arachidonic acid metab- Institute, Vienna, Austria) and was diluted in olism, PAF, or Csa and production of the sterile PBS. Original stock cytokine prepactivity can be inhibited by actinomycin D or arations contained endotoxin levels below dexamethasone (Watson et al. i988b). O.OOI E.U./pmol cytokine. Agents which stimulate PMNs are generally potent inducers of increased vascular perProduction of PMN-stimulating activity (PSA) meability in vivo (Wedmore & Williams I98I). To further characterize the inflam- Human synovial cells were obtained by collagenase and trypsin treatment of synomatory activity of PSA-containing supernavial tissue from arthritic patients undergoing tant, we have examined its ability to increase vascular permeability and cause PMN ac- corrective surgery and cultured in DMEM cumulation in rabbit skin, and compared its with io% HIFCS (Dayer et al. 1976). Subcultures (predominantly fibroblasts) were preactivities with those of human recombinant IL-I, tumour necrosis factor (TNFa) and pared by harvesting cells with trypsin-EDTA granulocyte-macrophage colony-stimulat- and seeded into new flasks at 3 x I04 cells/ ml. In the experiments described, culture ing factor (GM-CSF). medium from confluent 2nd-5th passage subcultures (i.2 X I05 cells/cm2) were Materials and methods removed by aspiration and replaced with DMEM plus 5% HIFCS together with IL-ia Materials (28 pM,), and cultures returned to the incu125I-Human serum albumin (1 25I-HSA) was bator for 24 h. After the incubation period PSA-containing supernatants were collected purchased from Amersham International, and stored at - 20°C until assay. PGE2 levels Bucks. Dulbecco's modified Eagle's medium in such supernatant were approximately I 5 (DMEM, with 25 mM HEPES and supplemented with penicillin-streptomycin, I00 pmol/o. i ml. Supernatant from synovial cell iu/ml), heat-inactivated foetal calf serum cultures not exposed to IL-i was also col(HIFCS) and sterile phosphate-buffered saline lected and used as a control. (PBS) were purchased from Gibco, Paisley, Scotland. Bovine serum albumin (BSA, Measurement of vascular permeability in rabbit essentially fatty acid free), prostaglandin E2 skin (PGE2) and bradykinin were purchased from Sigma, Poole, Dorset. Alphaxalone and Increased vascular permeability was alphadolone acetate (Saffan) was purchased assessed in rabbit skin by the measurement of the dermal accumulation of intravenously from Glaxovet, Uxbridge, Middlesex. administered 125I-HSA (Wedmore & Williams I 98 I). The dorsal skin of New Zealand Recombinant human cytokine preparations White rabbits (males, 2.5-4.o kg) was shaved I 6-20 h before the experiment. IL-I x (2.I X I0O7 lymphocyte activation facAnimals were anaesthetized with alphaxator units/mg); IL-I# (5 x IO7 U/mg); and
Cytokine-induced inflammation in rabbit skin lone and alphadolone acetate (4.5 and I.5 mg/kg initially), injection sites marked out, and test agents or controls (o. i ml/site) injected intradermally (i.d.) with 27 gauge hypodermic syringes according to a balanced site plan, with six replicates of up to 12 agents per animal. Thirty minutes before the end of the indicated time periods, '251-HSA (2.5 jCi/kg) was injected intravenously (i.v.) immediately followed by PGE2 (300 pmol/ site i.d. in o.i ml) at all sites. This superimposed PGE2 injection was given because an increase in vascular permeability alone is a poor stimulus of plasma extravasation (Wed-
30
95
more & Williams, I98I) and detection of increased permeability is facilitated in the presence of a vasodilator by increasing blood flow to the treated site (Williams & Peck, 1977). The PGE2 was always given during the monitoring period of the experiment to negate the effects of its inactivation in the skin before the measurement of plasma accumulation. In order to monitor permeability-increasing effects in the 30 min immediately after administration, this protocol was modified so that animals were first injected with 1251-HSA i.v. and then a mixture of test agents and PGE2 injected i.d. In
a
^ 20 E
,10
b .(D 40 a)
030 .
I T
I-I
**
.C 20 a,
(D10 -----------------------
a)
(
o
%.,0
240 120 180 60 Time after i.d. injection (min)
4 300
Fig. i. Effect of IL-ioa on vascular permeability and PMN accumulation in rabbit skin. (a) Plasma accumulation in rabbit skin at different times after i.d. injection of IL-ia (84 fmol/site, solid symbols) or vehicle, BSA (0.25 % in PBS, open symbols). Plasma accumulation in the presence of superimposed PGE, (300 pmol/site) was monitored over the 30 min preceding the indicated time points by the accumulation of i.v. administered 1 2 I-HSA. Points indicate mean ± s.e.m. pl plasma/site from at least four experiments. (b) Time-course of PMN accumulation in rabbit skin following i.d. injection of IL- i (solid symbols) or BSA (open symbols). After counting for radioactivity, skin samples were fixed, sectioned (7 gm thick) and stained. PMN were counted in successive high-power fields between the outer dermis and subcutaneous muscle. Points indicate mean ± s.e.m. PMN/high-power field of sections from four experiments. *, Significantly increased compared with BSA-treated sites, P
