Tachykinin receptors on human monocytes: their involvement in rheumatoid arthrit is
S. Brune l lesch i , ~ G. Bordin , a D. C o l a n g e l o J I. V iano 1
~Department of Medical Sciences, University of Turin, Faculty of Medicine, Novara 211 Division of Medicine, Az. Ospedaliera 'Maggiore della Carit&', 28100 - Novara, (Italy)
Summary Three types of tachykinin receptors, namely NK 1, NK 2 and NK 3, are known to preferentially interact with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), respectively. Experimental evidence indicates that SP and NKA modulate the activity of inflammatory and immune cells, including mononuclear ones. This study evaluated the effects of mammalian tachykinins and selective tachykinin agonists and antagonists on human monocytes isolated from healthy donors: SP, NKA and NKB all evoked a dose-dependent superoxide anion (02-) production and the NK 2 selective agonist [I3-AlaS]-NKA(4-10) induced a full response. The NK s selective agonist senktide was inactive, while the NK1 selective agonists septide and [Sar9Met(O2)lqSp displayed some effects. These results indicate that NK 2 and also some NK~ receptors are present in monocytes isolated from healthy donors. The role of tachykinin receptor activation in rheumatoid arthritis was also investigated, by measuring 02- production and TNF-cz mRNA expression in monocytes isolated from rheumatoid patients. Tachykinins enhanced the expression of this cytokine in both control and rheumatoid monocytes and NK 2 receptor stimulation was shown to trigger an enhanced respiratory burst in monocytes from rheumatoid patients. In conclusion, these results indicate that NK 2 and NK~ receptors are present on human monocytes, the former being preferentially involved in rheumatoid arthritis.
INTRODUCTION
Considerable interest has developed recently in evaluat- ing the effects of mammalian tachykinins - a family of peptides with the same C-terminal sequence Phe-X-Gly- Leu-Met-NH2, - on the immune system. Three types of tachykinin receptors, namely NKj, NK 2 and NK3, are known to preferentially interact with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), respec- tively. 1 SP, which is synthesized in primary afferent neu- rons and released from the terminals in response to different stimuli, is known to largely contribute to the local control of the immune response. It stimulates lym- phocyte proliferation and interacts with specific recep- tors on human T lymphocytes and cultured human IM-9 lymphoblasts, potentiates IgA and igM antibody produc-
Received 17 November 1997 Accepted 12 January 1998
Correspondence to: S. Brunelleschi, Department of Medical Sciences, Faculty of Medicine, University of Turin, Via Solaroli 17,2810O Novara, Italy. Tel: 0321-660648/660639, Fax: 0321-620421, E-mail: [email protected]
tion in vitro and in vivo, reduces IgE antibody-forming cell responses in vivo and in vitro, induces eosinophil activation and modulates antigen-induced IFN 7 produc- tion in murine schistosomiasis? -z Moreover, SP is known to degranulate mast cells, to modulate human neutrophil activity and to stimulate guinea pig peritoneal macro- phages as well as rodent and human alveolar macro- phages. 8-1~ Mononuclear phagocytes, either circulating blood monocytes or tissue macrophages, participate in host defense responses through their capacity to present antigens, to produce oxy-radicals and to release different soluble mediators (cytokines, eicosanoids, lysosomal enzymes). Lotz et all6 demonstrated that SP and NKA, as well as the C-terminus peptide SP(4-11), acting at low concentrations (maximal effects at 10 nM), induce the release of inflammatory cytokines (such as IL-1, TNF~ and IL-6) from human blood monocytes; more recently, however, monocyte activation has been reported only at high micromolar concentrations of SP 17 and a non-neu- rokinin substance P receptor has been described in these cells. ~z~s In human peripheral blood mononuclear cells, NKA and the truncated peptide NKA(4-10) produce a
215
216 Brunelleschi et al
dose-dependent (1 fM-0.1 riM) increase of the mitogenic response to l gg/ml phytohemagglutinin (PHA). 19 Moreover, alveolar macrophages, which are largely derived from circulating blood monocytes isolated from guinea pigs and healthy human smokers present mainly tachykinin NK 2 receptors.12,~
With these observations in mind, a first aim of the pre- sent study was to evaluate, by means of selective tachykinin receptor agonists and antagonists, the pres- ence, if any, of tachykinin receptors on human blood monocytes isolated from healthy donors.
Rheumatoid arthritis (RA) is a chronic systemic disease of unknown cause, mainly characterized by persistent inflammatory synovitis (usually involving peripheral joints in a symmetric distribution), abnormal cellular and humoral immune responses - accumulation of T lympho- cytes, particularly those expressing the CD4+, CDw29 memory phenotype of the helper-inducer subset, in the synovium; autoantibodies, especially rheumatoid factors and antibodies against collagen type II, are often present and synovial hyperplasia - characterized by proliferating synoviocytes and large numbers of infiltrating macrophages. 2° Experimental evidence supports the involvement of neuropeptides in the pathogenesis and development of RA. Elevated levels of tachykinin-like activity have been detected in joint fluids from patients with rheumatic inflammatory disease, 21 SP has been shown to contribute to the severity of experimental arthritis and to stimulate PGE 2 and collagenase release from rheumatoid synoviocytes. 22,23 Moreover, tachykinin NK 1 receptors are present in human synovium to mediate pro-inflammatory actions of locally released SP 24 and a loss of immunoreactive fibers for protein gene product 9.5, the C-flanking peptide of neuropeptide Y, substance P and calcitonin gene-related peptide has been observed in superficial synovium isolated from rheumatoid patients. 25
Tumor necrosis factor (TNF) is a pro-inflammatory cytokine involved in the pathogenesis of RA: it induces the proliferation of synoviocytes and enhances their pro- duction of prostaglandins, metalloproteinases and cytokines. 2¢27 TNF concentrations are increased in the synovial fluid of patients with active RA 28 and TNF antag- onists have been shown to be effective in the treatment of RA59,3° So, the study also aimed to evaluate tachykinin effects on circulating blood monocytes obtained from newly diagnosed RA patients and to investigate whether or not they could modulate TNF-cz expression.
MATERIALS AND METHODS
Isolation of peripheral blood monocytes
Peripheral blood monocytes were isolated from heparinized venous blood (30-40ml) by standard
techniques of dextran sedimentation (dextran T-500, Pharmacia) and Ficoll-Paque (density = 1.077g/cm3; Pharmacia) gradient centrffugation (400 x g 30 min) and recovered by thin suction at the interface. Cells were then washed twice with warm balanced phosphate- buffered saline ((PBS, pH Z4; Sigma) and resuspended at 1-2 x 107/ml in RPMI 1640 medium (Sigma), supple- mented with 5% heat-inactivated fetal calf serum (Sigma), 2 mM glutamine (Sigma), 50 btg/ml streptomicin and 5 U/ml penicillin (Sigma). Cell viability, as assessed by trypan blue dye exclusion, was > 98%. Purified mono- cyte populations were obtained by adhesion and assessed with the pan-macrophage monoclonal antibody anti-CD14 (Becton Dickinson). Briefly, 100 gl of cell sus- pension were plated in six-well tissue culture plates (35 mm diameter, Costar) and allowed to adhere for 90 rain at 37°C in a humidified atmosphere containing 5% CO 2. The non-adherent cells (mainly lymphocytes) were removed by three gentle washings with warm PBS. In some experiments, peripheral blood monocytes were iso- lated from patients with newly diagnosed rheumatoid arthritis, according to the 1987 revised criteria of the American Rheumatism Association (see below).
Superoxide anion production
Adherent monocytes (0.6-1 × 10ffdish) were washed twice with PBS and then challenged with increasing con- centrations of substance P (SP), neurokinin A (NKA), neurokinin B (NKB) or selective tachykinin receptor ago- nists ([[3-AlaS]-NKA(4-10), NK 2 agonist; [SargMet(Og)n]SP and septide, NK 1 agonists; senktide, NK 3 agonist) for 30 min. The effects of tachykinins were compared with those evoked by the bacterial peptide N-formylme- thionyl-leucyl-phenylalanine (FMLP) and the direct pro- tein kinase C activator, phorbol 12-myristate 13-acetate (PMA). In some experiments cells were pretreated for 15 min at 37°C with a cocktail of inhibitors: captopril, thior- phan and bestatin, all at 1 gM. This concentration has been shown to block tachykinin-degrading enzymes. 15 In the experiments with tachykinin receptor antagonists (CP 96, 345, NK~ antagonist; MEN 10,627, NK 2 antago- nist), monocytes were preincubated for lOmin with these drugs and then challenged with tachykinins.
Superoxide anion (02-) production was evaluated by superoxide dismutase-inhibitable cytochrome C reduc- tion, the absorbance changes being recorded at 550 nm in a Beckman spectrophotometer, and expressed as nmol cytochrome C reduced/10 ~ monocytes/30 min.~5
Experiments were performed in duplicate and control values (e.g. basal 02 - production) were subtracted from all determinations. All results are expressed as means + SEM. Statistical analysis was performed by Student's t- test.
Tachykinin receptors on monocytes: in rheumatoid arthritis 217
RNA extraction and Northern blot analysis
Plates containing 5 x 10 ~ monocytes, from both control or rheumatoid patients, were stimulated by a fixed con- centration (10 -7 M) of PMA, SP, NKA or the NK 2 agonist [[~-AlaS]-NKA(4-10) for 30 min at 37°C in a humidified atmosphere containing 5% CO 2. For the preparation of total cellular RNA, monocytes were washed twice with Hanks' balanced salt solution and immediately lysed. RNA was extracted and collected using RNA Fast solu- tion (Molecular Systems, USA), according to the manu- facturer's instructions. For Northern blotting, 10-20 ~g of heat-denatured total RNA were loaded in each line and separated on a 1% agarose, 0.6% formalde- hyde/MOPS denaturating gel, transferred to a nylon membrane (Hybond N+; Amersham, UK) and immobi- lized by baking at 80°C. Probes, end-labeled with [y32p]ATP and able to specifically recognize TNF-cz mRNA, were designed on the published sequences and verified with the EMBL Fasta and Word Search programs. The prehybridizations were performed with the QuikHyb solution (Stratagene) with the addition of 100 ~g/ml salmon testis DNA (Sigma, USA). Blots were stripped and reprobed for human [3-actin mRNA, used as control for RNA loading. Expression levels and densito- metry were determined with a Molecular Imager (Model GS250, BioRad, USA).
Patients
Eight patients, two males and six females, aged between 48 and 70 years, with newly diagnosed rheumatoid arthritis were studied. Diagnosis was done according to the 1987 revised criteria for classification of RA (four of the seven criteria listed below are required: morning stiff- ness, arthritis of three or more joint areas, arthritis of hand joints, symmetric arthritis, rheumatoid nodules, serum rheumatoid factor, radiographic changes). All patients gave their informed consent and received no therapy at the time of the study. The study and the research protocol were approved by the Ethical Committees of the University of Turin and Azienda Ospedaliera 'Maggiore della Carit~' of Novara.
Chemicals
The compounds used and their sources were as follows: SP, NKA, NKB, [[~-MaSI-NKA(4-10), [SargMet(O2)~]SP, senktide, septide, CP 96,345 and bestatin (Peninsula, UI0; thiorphan, captopril, superoxide dismutase, cytochrome C type VI, FMLP, PBS and RPMI 1640 (Sigma, UK). MEN 10,627 was a kind gift from Dr C.A. Maggi, Menarini Laboratories, Firenze, Italy.
RESULTS
Effects of mammalian tachykinins on superoxide anion production by human monocytes
In the concentration range 10-13-10 -~ M, the three mam- malian tachykinins dose-dependently evoked 02- pro- duction by blood monocytes isolated from healthy donors (Fig. 1). As depicted in Figure 1A, maxdmal activa- tion (3.02 +_ 0.3 nmol cytochrome C reduced/10 e mono- cytes/30 min; n = 8) by SP was observed at 1 gM, EDs0 being 0.01 nM. In the presence of a cocktail of inhibitors (thiorphan, captopril, bestatin, all at 1 ~tM) of tachykinin- degrading enzymes, SP effects were ,,significantly enhanced for concentrations up to 10 -9 M and EDso, under these conditions, was about 0.3 pM (Fig. 1A). NKA, which preferentially interacts with tachykinin NK 2 recep- tors, dose-dependently evoked 02 - production from human monocytes: ED~o value amounted to 9 pM and maximal activation (at 1 pM) was 3.36 + 0.3nmol cytochrome C reduced/lO ~ monocytes/3Omin (n = 8) (Fig. 1B). NKA-evoked respiratory burst was also potenti- ated when monocytes were pretreated with the cocktail of enzyme inhibitors, EDs0 being 1.5 pM (Fig. 1B). NKB, at concentrations ranging from 10 -53 M to 10 -~ M, induced 02- production from human monocytes with an EDs0 value of 25 pM: its effects were also enhanced (EDso -- 0.3 pM) in the presence of thiorphan, captopril and bestatin (Fig. 1C). Maximal activation evoked by NKB (2.3 + 0.3 nmol cytochrome C reduced/lO ~ monocytes/30 rain at 1 ~tM; n = 8) was smaller than that induced by equal con- centrations of the two other mammalian tachykinins.
When human monocytes were challenged by FMLP 1 ~M or PMA 1 ~M, 02- production was 6.5 + 2 and 22.5 + 3.4 nmol cytochrome C reduced/10 ~ monocytes/30 rain (n = 8; data not shown), respectively.
Effects of selective tachykinin receptor agonists on superoxide anion production by human monocytes
In the concentration range 10 -~3 M-10 -6 M, the selective NK 2 receptor agonist [[~-MaS]-NKA(4 - 10) evoked 02- pro- duction by human monocytes with maximal ,effects (2.88 + 0.3nmol cytochrome C reduced/10 ~ monocytes/30 min; n -- 5) at 1 ~tM and an EDso value of 17 pM (Fig. 2). The NK~ selective agonist [SargMet(O2)~l]SP, although less active and potent than the NK 2 selective agonist, evoked a significant respiratory burst from human monocytes: maximal effects (1.15 + 0.17nmol cytochrome C reduced/106 monocytes/30 rain; n = 5) were achieved at 1 ~M and EDso value was 2 nM (Fig. 2). On the contrary, the NK 3 selective agonist senktide was almost ineffective (Fig. 2). Based on the observation that septide ([pGlu, Prog]SP(6-11) showed reduced affinity on the NK 1 site in
binding studies in guinea pig ileum membranes, while maintaining full biological activity in guinea pig ileum, a new 'septide receptor' had been proposed? 1,32 As shown in Figure 3, which compares the effects of SP with those evoked by NK1 selective agonists, septide dose-depen- dently stimulated 02 production by human monocytes: EDso value was about 5 nM, maximal activation (2.3 + 0.5 nmol cytochrome C reduced/10 ~ monocytes/30 min; n = 5) was achieved at septide 1 ~tM (Fig. 3).
Effects of se lec t ive t a c h y k i n i n - r e c e p t o r a n t a g o n i s t s on
s u p e r o x i d e an ion product ion by h u m a n m o n o c y t e s
The non-peptide NK 1 selective antagonist CP96,345 at 1 nM competitively antagonized the effects of SP (Fig. 4). The dose-response curve for SP was shifted to the right (about two orders of magnitude; Fig. 4), as was that for septide (data not shown). In the presence of the competi- tive NK 2 receptor antagonist MEN 10,627 at 1 nM, con- centration-effect curves for NKA and [[3-AlaS]-NKA(4-10) were shifted to the right (Fig. 5A, B, respectively), by about two orders of magnitude.
B.C. M 61 70 + M.M. F 48 45 - B.L. F 50 27 - R.R. F 51 42 + B.G. M 68 80 + P.E. F 57 15 - T.D. F 51 34 + R.R. F 70 > 120 +
Patients were diagnosed for rheumatoid arthritis according to the 1987 revised criteria for the classification of rheumatoid arthritis. All patients had a newly diagnosed rheumatoid arthritis and had received no therapy at the time of the study.
Effects of tachykinins on superoxide anion production by human monocytes isolated from rheumatoid patients
In keeping with the aims of this investigation, human monocytes were also isolated from eight newly diag- nosed rheumatoid patients, whose characteristics are reported in Table 1.
SP dose-dependently evoked 02- production by rheumatoid monocytes with no significant variation, either in the amount of maximal activation or in its activ- ity, as compared with control monocytes (EDs0 = 0.01 nM in control and 0.012 nM in rheumatoid) (Fig. 6A).
The dose-response curve for NKA was linearly shifted upwards in monocytes from rheumatoid patients as com- pared with controls (EDso --- 0.87 pM in rheumatoid and 9 pM in control cells) with no significant variation in the amount of maximal activation (Fig. 6B), while the dose- response curve for the NK 2 selective agonist [[~- AlaB]NKA(4-10) was shifted upwards with a significant enhancement in the amount of 02- production in mono- cytes from rheumatoid patients (Fig. 6C). ED5o values for the NK a selective agonist were 17 pM in control and 7 pM in rheumatoid patients (Fig. 6C).
Results obtained with NKB indicate no appreciable variation between control and rheumatoid patients (data not shown).
Effects of tachykinins on TNF-c~ mRNA expression in monocytes from controls or patients with rheumatoid arthritis
Because of the key role played by TNF-~z in the patho- genesis and development of rheumatoid arthritis, this study evaluated the effects of tachykinins and PMA on TNF-c~ mRNA expression in monocytes obtained from either controls or rheumatoid patients, Monocytes were challenged for 30 min (the same time scale as that in experiments measuring superoxide anion production)
Tachykinin receptors on monocytes: in rheumatoid arthritis 221
Normal Rheumatoid Arthritis
j g e
~-- TNF-o~
~ acfin
Fig. 7 Effects of PMA and tachykinins on TNF-c~ mRNA levels in monocytes from control or rheumatoid patients. Human monocytes were challenged for 30 min with buffer, PMA, SP, NKA or [~-AlaS] - NKA(4-10). See Materials and Methods for further details.
with a fixed concentration (0.1 gM, the near maximal dose tested in experiments measuring superoxide anion production) of tachykinins or PMA. As depicted in Figure 7, an enhanced TNF-~ mRNA expression (2-4-fold) was observed in untreated monocytes obtained from r]heumatoid patients as compared with healthy donors. PMA, SP, NKA and [[~-AlaS]-NKA(4-10) enhanced TNF-~z mRNA about two-fold, with no significant difference between healthy donors and rheumatoid patients. Results presented in Figure 7 are representative of three other experiments.
D I S C U S S I O N
This study demonstrates that the neurokinins SP, NKA and NKB induce 0 2- production in monocytes obtained from healthy donors or rheumatoid patients.
With regard to SP, the study demonstrated that it acted dose-dependently just at low doses, its effects being com- petitively antagonized by CP 96,345 (a selective non-pep- tide NK~ antagonist) and enhanced in the presence of a cocktail of inhibitors of those enzymes (neutral endopep- tidase, angiotensin-converting enzyme, aminopeptidase) involved in its degradation. It is well known that neutral endopeptidase is involved in the catabolism of all neu- rokinins, while angiotensin-converting enzyme preferen- tially degrades SP? Moreover, SP effects were reproduced, although to a lesser extent, by the NK~ selective agonists [Sarg,Met(O2)~qSP and septide. As a result of different experimental approaches, the binding of SP requires the
NH 2 terminal of the tachykinin, whereas the biological activity does not. Septide, which lacks the NH 2 terminal, binds to receptors with a reduced strength and, as a general rule of all partial sequences, interacts with a smaller number of receptor sites (as compared to large agonists) and dissociates faster? So, this may explain the activity of septide in the present study, although it was less potent than that of the large agonist SR These data are in very good agreement with the study by Lotz et al,~6 indicating that SP, with a maximum at 10-100nM, evokes TNF-c~, IL-1 and IL-6 release from human mono- cytes, but the results are at variance with those more recently reported by a Dutch group, lz~s As reported by Kavelaars et al,~z SP stimulated IL-6 secretion by human monocytes only at concentrations (30-100 gM) so high that a classical receptor interaction cannot be envisaged. SP effects were not reproduced by a n N K 1 selective ago- nist and were even more pronounced with [D-Pro2,D - TrpZg]SP, a n NK I receptor antagonist: so, the authors stated that SP effects on human monocytes were afforded by a non-neurokinin SP receptor, functionally coupled to a G~ protein) 7 Activation of this SP receptor leads to stim- ulation of mitogen-activated protein (MAP) kinase and phospholipase D, as well as a rapid rise in cytosolic cal- cium: all effects are evidenced at high micromolar con- centrations and are blocked by monocyte pretreatment with pertussis toxin. ~7
By performing binding studies in human monocytes, Jeurissen et al~s reported that this novel 'atypical' SP receptor presents a K d of 2.2 x 10 -7 M and a Br~ ~ of 4.7 pmol/mg membrane protein. However, it remains to be explained (the Dutch group did not attempt this) why SP stimulated IL-1 and IL-6 secretion in the concentration range 0.1 nM-1 gM, 16 but acted only at 30-100 gM on IL- &stimulated release in the study by Kavelaars et al) 7
It is also worth noting that in human bone marrow mononuclear cells, SP induces the production of both IL- 3 and GM-CSF, through the release of IL-1 and IL-6: this induction requires de novo synthesis, is blocked by an NK~ receptor antagonist, is dependent on the C-terminus moiety and happens at an optimal concentration of 10-" M SP. 33
Morevoer, the Dutch group did not evaluate NKA effects, whereas Lotz et aP 6 and this study did: NKA was even more potent than SP in both studies. This study also used [~-AlaS]NKA(4-10), a selective NK 2 receptor agonist, which displayed full biological activity. Its effects, as well as those evoked by NKA, were competitively antagonized by MEN 10,627, an NI~ selective antagonist, thus con- firming the presence of NK 2 receptors on human mono- cytes. The situation depicted here - prevalence of NK 2 receptors and minor participation of NK~ receptors - is strongly reminiscent of that previously documented in guinea pig alveolar macrophages? 2 To our knowledge,
while many different groups have investigated the effects of SP on different inflammatory and immunocompetent cells, less attention has been paid to the evaluation of the effects of the two other neurokinins, NKA and NKB.
Casini et al, 19 while evaluating the mitogenic response on human peripheral blood mononuclear cells (a non- homogenous population, mainly composed of B and T lymphocytes, monocytes and macrophages), showed that NKA and its fragment NKA(4-10) - which is endowed with a better NK 2 selectivity, have no direct stimulation per se - produced a concentration-dependent (1 fM-10 pM) potentiation of the response to 1 pg/ml PHA (a well- known mitogen for these cells), while having no effect on the response to 25 pg/ml PHA.
Kimball et aP 4 found that SP, as well as NKA and NKB, induced IL-1 production from the mouse macrophage cell line P388D1 and discussed their possible relevance to arthritic disease. This fits very well with the second aim of the present investigation, that was to evaluate the effects of mammalian tachykinins and selective agonists on monocytes isolated from newly diagnosed rheuma- toid patients. It has long been demonstrated that SP largely contributes to the severity of experimental arthri- tis, 22 induces PGE 2 and collagenase release from human synoviocytes in vitro and evokes synoviocyte prolifera- tion, thus enhancing the formation of pannusY Walsh et a124 demonstrated the presence of NK 1 receptors in rat and human synovium, which may participate in inflam- matory actions of locally released SP, while immunohis- tochemical studies have pointed to a reduction of SP-containing nerves in the superficial layers of chroni- cally inflamed synovium in human rheumatoid arthri- tis 25. Loss of neuropeptide immunoreactivity might be due to their local release, as proposed by Grondblad et al,35 or might result from synovial tissue hyperplasia without proliferation of new fibres and/or degeneration of pre-existing nervesY The results of the present study indicate that SP, NKA and the NK 2 selective agonist [~- Ala~]NKA(4-10) dose-dependently evoke 02- production from rheumatoid monocytes. The eight patients involved in this study were newly diagnosed and received no ther- apy at the time of blood sample collection. However, while no difference between rheumatoid and control monocytes was detected with SP, NK 2 receptor stimula- tion resulted in a greater activity. In fact both NKA and the NK 2 selective agonist induced an enhanced 02- pro- duction in monocytes obtained from rheumatoid patients as compared with controls, [[3-AlaS]NKA(4-10) being particularly effective, because the amount of the respiratory burst was more than doubled in the concen- tration range 0.1 nM - 1 ~tM. Moreover, by evaluating TNFcz mRNA expression in monocytes from both control and rheumatoid patients, it was possible to demonstrate a more than doubled expression of this cytokine in
untreated cells from rheumatoid patients and an enhanced expression after challenge with tachykinins. These results are in agreement with observations by Lotz et al, ~6 indicating the ability of NKA and SP to evoke, at low concentrations, TNFa release from human mono- cytes. However, further experiments, e.g. time-course experiments for cytokine expression (this study only tested a 30 min stimulation period, the same as the opti- mal time for 02 - production) and determinations of the amount of cytokine release, are needed to establish a clear role for neurokinin-cytokine cross-talk in RA.
In conclusion, these results indicate that NK 2 and NK 1 receptors are present on human monocytes and may par- ticipate in the development of autoimmune diseases, such as RA.
A C K N O W L E D G E M E N T S
We wish to thank Dr C.A. Maggi (Menarini Laboratories, Florence, Italy) for the kind gift of the NK 2 selective antagonist, MEN 10,62Z This work was supported in part by grants from MURST 60%, 1996, 199Z
REFERENCES
1. Regoli D, Boudon A, Fauchere J L. Receptors and antagonists for substance P and related peptides. Pharmacol Rev 1994; 46: 551-599.
2. Payan D G, Brewster D R, Goetzl E J. Specific stimulation of human T lymphocytes by substance P. J Immunol 1983; 131: 1613-1615.
3. Bost K L, Pascual D W, Substance P: a late-acting B-lymphocyte differentiation cofactor. Am J Physiol 1992; 262: C537-C545.
4. Payan D G, McGillis J P, Organist M L. Binding characteristics and affinity labelling of protein constituents of the human IM-9 lymphoblast receptor for substance P. J Biol Chem 1986; 261: 14321-14329.
5. Carucci Auci D L, Herrick C A, Durkin H G. Neuropeptide- mediated regulation of hapten-specific IgE responses in mice. I. Substance P-mediated isotype-specific suppression of BPO- specific IgE antibody-forming cell responses induced in vivo and in vitro. J Leukoc Biol 1995; 57:110-115.
6. Iwamoto I, Nakagawa N, Yamazaki H, Kimura A, Tomioka H, Yoshida S. Mechanism for substance P-induced activation of human neutrophils and eosinophfls. Reg Pept 1993; 46: 228-230.
Z Blum A M, Metwali A, Cook G, Mathew R C, Elliott D, Weinstock J V. Substance P modulates antigen-induced, IFN- 7 production in murine schistosomiasis mansoni. J Immunol 1993; t51: 225-233.
8. Mousli M, Bronner C, Bueb J L, Tschirhart E, Gies J P, Landry Y. Activation of rat peritoneal mast cells by substance P and mastoparan. J Pharmac Exp Ther 1989; 250: 329-335.
9. Bar-Shavit Z, Goldman R, Stabinsky Yet al. Enhancement of phagocytosis. A newly found activity of substance P residing in its N-terminal tetrapeptide sequence. Biochem Biophys Res Commun 1980; 94: 1445-1451.
10. Brunelleschi S, Tarli S, Giotti .4, Fantozzi R. Priming effects of mammalian tachykinins on human neutrophils. Life Sci 1991; 48: PL1-PL5.
Tachykinin receptors on monocytes: in rheumatoid arthritis 223
11. Hartung H P, Wolters K, Toyka K V. Substance P: binding properties and studies on cellular responses in guinea pig macrophages. J Immunol 1986; 136: 3856-3863.
12. Brunelleschi S, Vanni L, Ledda F, Giotti A, Maggi C A, Fantozzi R. Tachykinins activate guinea-pig alveolar macrophages; involvement of NK 2 and NK 1 receptors. Br J Pharmacol 1990; 100: 417-420.
13. Brunelleschi S, Parenti A, Ceni E, Giotti A, Fantozzi R. Enhanced responsiveness of ovalbumin-sensitized guinea-pig alveolar macrophages to tachykinins. BrJ Pharmacol 1992; 107: 964-969.
14. Brunelleschi S, Guidotto S, Tonso E, Viano I, Fantozzi R. Modulation by protein kinase C of the enhanced responsiveness to tachykinins in ovalbumin-sensitized guinea pig alveolar macrophages. Neuropeptides 1996; 30: 249-260.
15. Brunelleschi S, Guidotto S, Viano Ie t al. Tachykinin activation of human alveolar macrophages in tobacco smoke and sarcoidosis: a phenotypical and functional study. Neuropeptides 1996; 30: 456-464.
16. Lotz M, Vaughan J H, Carson D A. Effects of neuropeptides on production of inflammatory cytokines by human monocytes. Science 1988; 241: 1218-1221.
17. Kavelaars A, Broeke D, Jeurissen F et al. Activation of human monocytes via a non-neurokinin substance P receptor that is coupled to Gi protein, calcium, phospholipase D, MAP kinase, and IL-6 production. J Immunol 1994; 153:3691-3699.
18. Jeurissen F, Kavetaars A, Korstjens Me t al. Monocytes express a non-neurokinin substance P receptor that is functionally coupled to MAP kinase. J Immunol 1994; 152: 2987-2994.
19. Casini A, Geppetti P, Maggi C A, Surrenti C. Effects of calcitonin gene-related peptide (CGRP), neurokinin A and neurokinin A(4-10) on the mitogenic response of human peripheral blood mononuclear cells. Naunyn-Schmiedebergs Arch Pharmacol 1989; 339: 354-358.
20. Gay S, Gay R E, Koopman WJ. Molecular and cellular mechanisms of joint destruction in rheumatoid arthritis: two cellular mechanisms explain joint destruction? Ann Rheum Dis 1993; 52: $39-$4Z
21. Devillier P, Weill B J, Menkes C J, Pradefles P, Renoux M. Elevated levels of tachykinin-like activity in joint fluid from patients with rheumatic inflammatory diseases. N Engl J Med 1986; 314: 1323.
22. Levine J D, Clark R, Devor M, Helms C, Moskowitz M A, Basbaum A I. Intraneural substance P contributes to the severity of experimental arthritis. Science 1984; 226: 547-549.
23. Lotz M, Carson D & Vaughan J H. Substance P activation of rheumatoid synoviocytes: neural pathway in pathogenesis of arthritis. Science 1987; 235: 893-895.
24. Walsh D A, Salmon M, Mapp P I et al. Microvascular substance P binding to normal and inflamed rat and human synovium. J Pharmacol Exp Ther 1993; 267: 951-960.
25. Mapp P I, Kidd B L, Gibson SJ et al. Substance P-, calcitonin gene-related peptide- and C-flanking peptide of neuropeptide Y- immunoreactive fibres are present in normal synovinm but depleted in patients with rheumatoid arthritis. Neuroscience 1990; 37: 143-153.
26. Dayer J M, Beutler B, Ceranli A. Cachectin/tumor necrosis factor stimulates collagenase and prostaglandin E2 production by human synovial cells and dermal fibroblasts. J Exp Med 1985; 162: 2163-2168.
27. Alvaro-Garcia J M, Zvaifler N J, Firestein G S. Cytokines in chronic inflammatory arthritis. Mutual antagonism between interferon-gamma and tumor necrosis factor-alpha on HLA-DR expression, proliferation, collagenase production, and granulocyte macrophage colony-stimulating factor production by rheumatoid arthritis synoviocytes. J Clin Invest 1990; 86: 1790-1798.
28. Saxne T, Palladino M A Jr, Heinegard D, Talal N, Wollheim F A. Detection of tumor necrosis factor alpha but not tumor necrosis factor beta in rheumatoid arthritis synovial fluid and serum. Arthritis Rheum 1988; 31: 1041-1045.
29. Elliott M J, Maini R N, Feldmann M e t al. Randomized double- bind comparison of chimeric monoclonal antibody to tumor necrosis factor alpha (cA2) versus placebo in rheumatoid arthritis. Lancet 1994; 344:1105-1110.
30. Moreland L W, Baumgartner S W, Schiff M H et al. Treatment of rheumatoid arthritis with a recombinant human tumor necrosis factor receptor (p75).Fc fusion protein. N Engl J Med 1997; 337: 141-14Z
31. Petitet F, Saffroy M, Torrens Yet al. Possible existence of a new tachykinin receptor subtype in the guinea pig ileum. Peptides 1992; 13: 383-388.
32. Maggi C A, Patacchini R, Meini S, Giuliani S. Evidence for the presence of a septide-sensitive tachykinin receptor in the circular smooth muscle of the guinea pig ileum. Eur J Pharmacol 1993; 235:309-311.
33. Rameshwar P, Ganea D, Gascon P. Induction of IL-3 and granulocyte-macrophage colony-stimulating factor by substance P in bone marrow cells is partially mediated through the release of IL-1 and IL-6. J Immunol 1994; 152: 4044-4054.
34. Kimball E S, Persico E J, Vaught J L. Substance P, neurokinin A, and neurokinin B induce generation of IL-1 like activity in P388D 1 cells. Possible relevance to arthritic disease. J Immunol 1988; 141: 3564-3569.
35. Grondblad M, Konntinen Y T, Korkala O, Liesi P, Hukkanen M, Polal~ J M. Neuropeptides in synovium of patients with rheumatoid arthritis and osteoarthritis. J Rheumatol 1988; 15: 1807-1810.
36. Pereira J A, Carmo-Fonseca M. Peptide containing nerves in human synovium: immunohistochemical evidence for decreased innervation in rheumatoid arthritis. J Rheumatol 1990; 17: 1592-1599.
