The immunoregulatory abilities of polymorphonuclear neutrophils in the course of multiple sclerosis.

The polymorphonuclear neutrophils (PMN) possess sufficient potential to affect both immune response and inflammation, however it has not been yet described in the course of multiple sclerosis (MS). We have studied binding of fluorescein isothiocyanate (FITC)- stained TNF-alpha by PMN, the expression of CD11a, CD11b, and CD18 molecules of beta2-integrines and the expression of CD10 (neutral endopeptidase-NEP) and of CD13 (aminopeptidase N; APN) antigens on PMN in three different groups of MS patients. The control group included neurological patients (OND) with noninflammatory diseases. The obtained results have proved that during MS exacerbation and in the course of chronic progressive MS, PMN reveal several forms of preactivation, including significantly higher stained-TNF-alpha binding, higher expression of CD11b and CD18, as well as CD10 and CD13 antigens, in comparison with MS remission or OND. We suggest that the increased expression of these molecules on PMN of MS patients in exacerbation of the disease and to a lower degree in the course of CP-MS is a result of PMN priming, and directly prove the PMN involvement in the disease pathogenesis.


Introduction
Multiple sclerosis (MS) is a dise as e in w hich multifoc al inflammation and damage of the blood -brain barrier and myelin sheath are salie nt pathologic features. Overw he lming evidenc e demons trates that MS is a pre dominantly T-c ell and monoc yte/mac rophageme diated autoimmune disorde r. 1 ,2 Polymorphonuclear ne utrop hils (PMN) have not be en considere d as a ce ll population participating in it. 3 PMN can how e ver ex pre ss immunore gulatory abilities, that has not be en ye t de sc ribe d in the c ourse of MS. Activated in vitro PMN produc e a numbe r of immune mediators including cytokines like IL-1b , IL-4, IL-6, IL-8, IL-10, IL-12, TNF, TGF-b 1. 4 ,5 There fore the re gulatory functions of these ce lls may be postulate d in the c ourse of MS.
PMN in the pe ripheral blood (PB) of MS patients can be primed mainly by inflammatory cytokine s like IL-1, INF-g , or by TNF-a sec re te d by mononuclear ce lls. Hypothetically PMN priming or activation in PB of MS patients may dep end also on compleme nt (especially C5a) immunologic al c omplex es, or certain me tabolite s of the arachidonic ac id (LTB 4 , PAF).
Priming re sults in the enhanced ex p re ssion on PMN of re cep tors for chemokines and other chemotactic pep tide s, 6 priming also enhance s the ex pre ssion of CD11b/CD18 molecules on PMN c ell surface 7,8 w hich re sults in the indire ct ac tivation of PMN. 9,10 In the pre sente d pap er w e have suggeste d that PMN priming can be obse rved in MS patie nts p eriphe ral blood.

Patients and methods
Patients (34 total; 19 w omen and 15 men, aged 22-56 years) w ere se lecte d w ith a clinic ally definitive diagnosis of MS 1 1 and w ith Kurtzke Ex panded Disability Status Scale 12 scores 5 or few er and categorized as having the re lapsing-re mitting MS (RR-MS) course for at least 5 years but curre ntly in re mission (REM) (n =13), as having either sec ondary CP-MS for at least 2 ye ars (n =13) or RR-MS and currently ex pe riencing clinical ex ace rbation (REL) of the disease (n = 12). Ex acerbations w ere defined as the appearance of new symptoms or significant w orsening of the old ones, attributabl e to MS, for at least 24 hours w ithout any fever.
The control group c onsiste d of patients w ith other ne urologic al diseas es (OND) (14 total; nine w omen and five men, aged from 24 to 37 ye ars), including those w ith vasomotor headache (n = 8) and ischialgia (n = 6).

Sample collection
The studies w e re performed on PMN of the p eripheral venous blood, c ollec te d into heparin-containin g tubes (10 U/ml).

TNF labelling with fluorescein isothiocyanate (FITC)
The synthesis and biological analysis of the TNF molecules w ere pe rforme d at the Departme nt of Bioorganic Chemistry, Lódź, Poland (Pate nt No. 168858). TNF staining w as done w ith FITC (Serva), according to Shirakaw a e t a l. 13 100 mg of FITC and 100 m l of 0.2 M carbonate buffe r (pH = 9.2) w ere added to 400 m g of TNF, dilute d in 400 m l of PBS and then, incubated for 6 h at 4°C. The labelled TNF w as se parated from the non-labe lle d w ith FITC by gel filtration on a 3.5 cm 3 column, filled w ith Sep hadex G-25. TNF labelled FITC (TNF-FITC) w as ste rilize d by filtration, using a 0.22 m m filter and then, store d at 4°C. The staining e ffic ie nc y w as me as ure d, taking into account the absorbe ncy at 280 nm, in comparison w ith the absorbenc y at 495 nm. The inve stigations w ere performe d w ithin 10 days after the TNF labe lling due to re latively low stability of the labe lling.

Evaluation of TNF-FITC binding to PMN
One hundre d m l of the w hole blood w as inc ubate d w ith TNF-FITC at the conc entration of 1000 ng/ml for 2 h at 4°C. The erythroc ytes w e re lysed, using 1 ml of FACS Lysing Solution (Bec ton Dickinson) for 15 min at room te mpe rature. Ce lls w e re ce ntrifuge d for 5 min at 400 3 g , the n w ashed w ith 3 ml of PBS and suspe nded in 100 m l of PBS. The fluore sce nce intensity of PMN w as measured using a flow c ytometry FACSc an (Be cton Dickinson) and Lysis II softw are. Each measureme nt w as re peated four times and pre sente d here as a me an fluore sce nce inte nsity (MFI). Spe cificity of the binding of TNF-FITC to PMN w as evaluated in blocking ex pe rime nt by pre inc ubation of the PMN w ith non-staine d TNF in ex c ess for 45 min be fore TNF-FITC binding (Fig. 1).
Evaluation of the expression of CD11a, CD11b and CD18 molecules of LFA-1 and Mac-1 intregine and CD10 and CD13 Ag of neutral endopeptidase (NEP) and aminopeptidase N (APN) on PMN The dete rmination of molecule ex pre ssions on the surface of PMN w as p erformed in the w hole blood. Te chniques, gene rally use d for immunofluore sce nt labelling of the c ells in w hole blood c ollec te d on he parin, w ere applie d. One hundred m l of blood w as mix e d and inc ubated at room te mperature w ith appropriate quantitie s of monoclonal antib odies, provide d by Dako (Denmark). CD11a, CD11b and CD18 antib odies w e re used. A double-ste p staining proce dure w as used for the e valuation of CD10 and CD13 Ag ex pre ssion. Mouse immunoglobulins anti-CD10 and CD13 (Dako) w e re use d as a first ste p. Rabbit anti-mouse IgG polyclonal immunoglobulins G staine d w ith R-phycoerythrin w e re use d as a sec ondary antib ody. Mouse IgG2a, stained w ith RPE, w as used as a ne gative control. Erythroc ytes w ere eliminated by an addition of lysing solution (Be cton Dickinson) into the blood samples. Afte r a short time of inc ubation and rinsing, the cells w ere suspe nded in physiological buffe re d saline (PBS). FACscan flow cytome te r w ith a 488 nm argon lase r (Becton Dickinson) and Lysis II softw are w ere used. The re sults w ere ex pre ssed as the value s of MFI of the labe lle d surface antigens.

Results
The MFI of the PMN incubated w ith FITC-TNF w as highest in the group w ith ex ace rbation of RR-MS (REL) ( Table 1) and the value w as significantly higher (P< 0.01) compared w ith the other groups of MS patients and to OND. In the c ourse of CP-MS and in MS re mission (REM) the te ste d values w ere in the range observed in the control group (OND).
Ex pre ssion of CD10, CD13 Ag and CD11b/CD18 molecules on PMN w as signific antly inc re ased in REL (P< 0.05 and P< 0.01) c ompared w ith that ex amined in CP-MS, REM and OND (Table 1). CD11a ex pre ssion on PMN in REL w as the same as in CP-MS patients and in OND. In the c ourse of CP-MS only ex pre ssions of CD11b and CD18 but not CD10 and CD13 w ere significantly inc re as ed (P<0.01 and P<0.05) compared w ith OND. Ex pre ssions of CD11a, CD11b and CD10 w ere markedly diminishe d (P< 0.01 and P< 0.05) during re mission of MS compare d w ith the controls (Table 1).

Discussion
It has be en proved in pre vious studie s that there is a more inte nsive inflammatory re sp onse in ac ute ex ace rbations of RR-MS patie nts than in the c ourse of CP-MS. 2 The obtained data sugge st that re lapsingre mitting MS follow s c ycles of immunologic al activation as a re sult of Th-1 re sponse (ex ac erbation), w hich is then follow e d by a suppre ssor re sponse that dow nre gulates inflammation (re mission). 1 ,2 In CP-MS there is continuous low -grade inflammation w ith no obvious ex acerbations or re missions.
The involvement of TNF in immunopathologic al proce sse s in MS has alre ady be en know n for quite some time . 14 This cytokine is also one of the most pote nt priming factors for PMN in vitro . 7,1 5 We suggest that se rum TNF of patients w ith ac ute ex ace rbations of RR-MS or in low er de gree in the course of CP-MS can be the main p riming fac tor of PMN.
TNF in v itro stimulates the ex pre ssion of many of the PMN surface molecules. Simultaneously TNF stimulates its ow n re ce ptors ex pre ssion (TNF-R) on PMN. 16 This has be e n c onfirmed in our study as the inc re as ed TNF binding to PMN in the c ourse of ac ute ex ace rbations of MS. We have obse rved also the significant inc re as e of the CD10, CD13 Ag and CD11b/CD18 molecule ex pre ssion on PMN of MS patients, mainly in the c ourse of ex ac erbation compared w ith MS re mission and w ith OND groups. This may be a sign of priming of PMN in MS ex acerbation and to a low er degre e in the c ourse of CP-MS.
Podikoglou e t a l. 3 have show n that the typic al functions of PMN, like adhere nce , chemotax is, phagocytosis or bacte ricidal action have be en significantly diminishe d in PB of MS patients. The re sults that w e have obtained in our study correspond w ith Goto e t a l.. 1 7 In some e arlie r obse rvation s Aoki e t a l. 18 and Guarnieri e t a l. 1 9 have show n that the inc re as ed intrac ellular ne utral proteinase and medullasine c onc entrations can be the symptom of PMN activation in the blood of patie nts w ith acute ex ac erbation of MS.
PMN c ontain large amounts of proteinases, like NEP and APN, in their intra cellular granules. 2 0,21 The inc re as ed ex pre ssion on in v itro stimulated PMN is a re sult of rapid transloc ation of an intrac ellular pool to the c ell surface . 22 ,2 3 The incre as ed ex pre ssion of CD11b/CD18-Mac -1 mole cules probably re sults from their rapid shift from internal granule s to the surfac e of primed PMN. Such proce ss has not be en observe d in CD11a/CD18 mole cules of LFA-1 integrine ex pre ssion of in vitro prime d PMN. 24,25 In our studies w e have not notice d the inc re ased ex pre ssion of CD11a molecule on PMN of MS p atie nts w ith acute ex acerbations or in the c ourse of CP-MS. In our opinion, the se can sugge st PMN p riming in patients w ith active MS. Activated PMN c an produc e ox yge n and nitrogen spe cie s, some matrix de grading metalloprote inases and some c ytokine s. 5 ,26 Priming is a pre liminary e ve nt be fore re c eptor-induce d stimulation of PMN. It is the n a sign of these c ells' hype rre ac tivity. PMN priming features obse rved in the PB of patie nts w ith active MS sugge st that these c ells may participate in the MS immunopathology.
Po ly m o rp ho nu cle a r n e u tro p hils a nd m u ltiple s cle ro s is Mediators of Inflammation · Vol 7 · 1998 337