Expression of aminopeptidase N (APN) on peripheral blood mononuclear cells' surface as a marker of these cells' transendothelial migration properties in the course of multiple sclerosis.

CD13 Ag and CD11a, CD11b, CD18 molecule expression on peripheral blood mononuclear cells (PBMC) were studied as these cells' adherent or transendothelial migration properties in three different multiple sclerosis (MS) patients groups (total 38): with clinically active MS (acute exacerbation of MS and primary chronic progressive MS (CP-MS)) and with MS remission. The control group consisted of patients, suffering from other non-inflammatory neurological diseases (OND). The results of our study suggest that CD11a/CD18 molecules expression on PB lymphocytes, although higher on these cells' surface in the course of MS as compared to OND, does not differentiate clinical forms of MS. CD11a molecule expression on monocytes did not differ significantly in all tested MS patient groups in comparison to OND. Although the expression of CD11b/CD18 molecules on monocytes' surface shows their activation in the course of MS, it does not differentiate them either. However, CD13 Ag of APN expression on PBMC surface may be an immunological marker of MS clinical form. CD13 Ag expression may also be a sensitive marker of these cells' transendothelial migration properties.


Introduction
Multiple sclerosis (MS) is a primary demyelinating disease in w hich the blood -brain barrier (BBB) damage and mononucle ar ce lls' infiltration in ac tive plaques w ithin the c entral ne rvous system (CNS) re prese nt charac te ristic pathologic features. Inflammatory p roce sse s in CNS may be initiate d by ac tivated peripheral blood mononucle ar c ells (PBMC). 1 The y can migrate through BBB and enable de ve lopme nt of MS itself and its clinic al acute ex ac erbation. Such a dise ase evolution is base d on the animal model of allergic e nce phalomyelitis (EAE). In the course of clinically ac tive MS, the migration of PBMC through the BBB c ould be se condary to the local BBB disruption by inflammatory cytokines and ex trac ellular matrix de grading prote ase s produce d in p laque s in CNS, and thus w ithout the involveme nt of PBMC. 2 The se create a totally diffe re nt view on the disease immunopathology. The problem of w hether PMNL c an show adherent and transe ndothelial migration prope rties in the c ourse of clinic ally active MS could be solved indirec tly by e valuation of the b -integrin molec ule ex pression and ex trac ellular matrix degrading p rote ases produc tion by these cells. The molecule ex pre ssion on PBMC may also 'immunologic ally' diffe rentiate clinical pe riods of MS.

Material and methods
There w e re three groups of patients (total 38 ) age d 21-56 ye ars (mean 38.2 ± 10.1 years ) w ith clinic ally definite MS and w ith Ex panded Disability Status Scale sc ore s of 5 or low e r. Thirte en patie nts (se ven w omen and six me n ) w e re ex amined during the c ourse of acute MS ex ace rbation, 12 patie nts (se ven w omen and five men ) w ith primary chronic progre ssive MS (CP-MS) and 13 patients (six w omen and se ve n men ) during MS re mission. The control group (OND) consis te d of 14 p atie nts (seven w omen and se ven me n ), aged 22 -41 years (me an 31.3 ± 6.7 ye ars ), suffe ring from other ne urologic al diseases, like vasomotor he adache in 10 and epilepsy in four c ases.

Immunostaining and FACS analysis
Blood samples w e re obtained in the morning betw e en 8.00 and 9.00 a.m. by ve nipuncture. One hundred microlitres of blood w as mix ed and incubated at room te mperature w ith appropriate quantities of monoclonal antibodie s anti-CD11a, CD11b and CD18 p rovided by Dako (Denmark ). A doublestep staining procedure w as use d for the e valuation of CD13 Ag ex pression. Mouse immunoglobulin anti-

Short Communication
Mediators of Inflammation, 9, 45 -48 (2000 ) Mediators of Inflammation · Vol 8 · 1999 CD13 (Dako ) w as used as a first ste p. Rabbit antimouse polyclonal IgG stained w ith R-phyc oe rythrin w as use d as a se condary antibody. The rabbit IgG w as also used as a negative control. Erythrocytes w e re eliminate d by an addition of lysing solution (Be cton Dickinson ) into the blood samples. A FACsc an flow cytome te r w ith a 488-nm argon laser (Bec ton Dickinson ) and Lysis II softw are w ere used. The re sults w ere ex pressed as the values of me an fluoresc enc e inte nsity (MFI) of the labelled surfac e antigens. An ex amp le of CD13 Ag ex pression on peripheral blood lymphoc ytes is show n on Fig. 1.
The obtained re sults w e re analyse d, using Student's t-test and the Cochran -Cox rank te st.

Results
The ex pre ssion of CD11a /CD18 molecules of LFA-1 inte grine w as highe r on lymphoc ytes in the c ourse of clinically active MS (MS ac ute ex acerbation and in CP-MS) as w e ll as in MS remis sion c omparing to the OND group (Table 1 ). Among MS patients the ex p re ssion of LFA-1 mole cules on lymphoc ytes surface did not differ significantly.
CD11a molec ule ex pression on monocyte s did not differ signific antly in all the tested MS patie nt groups in comparison to the OND. The CD11b mole cule of Mac-1 integrine ex pression w as significantly higher on monocyte s in the course of MS acute ex ace rbations and in CP-MS c ompare d to MS remission and OND. CD11b ex pre ssion w as also highe r in the course of MS remission c ompared to the OND group. Similarly to CD11b mole cule ex pre ssion on monocytes, the diffe renc es w e re notice d in molec ule CD18 ex pre ssion.
Conve rse ly to the integrine s mole cule ex pression on PBMC, CD13 Ag ex pre ssion on lymphoc ytes and on monocyte s 'immunologically' differe ntiated clinical pe riods of MS. In the course of MS ac ute ex ace rbation and in CP-MS groups, it w as significantly enhance d in comparison to MS re mission and OND (Table 1 ).

Discussion
The mechanisms of PBMC migration through the BBB in the c ourse of MS are not ye t know n. In a se rie s of repe ated te sts, highe r b 1 and b 2 integrin molec ule s' ex pre ssion on CD4 + T-ce lls in ac ute MS ex ac erbation compared to MS remission w as not found. 3 Also, in our obse rvations LFA-1 and Mac-1 inte grine molecules' ex pre ssion on PBMC did not 'immunologic ally' differentiate transe ndothelial migration prop erties of PBMC in the course of MS. How e ve r, CD11a /CD18 molecules' ex pression on PB lymphoc yte s, in each group of MS patients, w as highe r than in OND patients. In our opinion this c ould sugge st an inc re ased pe rc entage of me mory c ells in the p eripheral blood of all the MS patients. This w as also show n in some other studie s. 4 It indire ctly suggests that circ ulating lymphocytes in MS are p re disposed to cross e ndothelial barriers. Also CD11b /CD18 molecules of Mac-1 inte grine ex pre ssion w ere higher on monocyte s' surface in the course of clinic ally ac tive MS compared to MS remission. This can be an effe ct of non-spe cific stimulation of CD11b /CD18 mole cules on the se c ells' surface s by immunologic al complex e s, or C3a and C5a. How e ve r Mac-1 has a much smaller effect on monoc yte adherenc e to endothelium than LFA-1. 5 Thus, it cannot be a marke r of monoc yte transe ndothelial migration in the c ourse of MS.
Conve rse ly to integrine molec ule s, CD13 Ag ex pre ssion on the PBMC surface c an be such a marker. CD13 Ag asse ssment is w idely applied in the e valuation of APN ex pression on PBMC surface . 6 APN is a zinc -de pende nt me talloproteinase . Similarly to 92 kDa ge latinase it hydrolyses the IV-type of collagen. 7 Therefore , it plays an important role in PBMC migration ac ross the blood vessels. Se ve ral studies revealed a crucial role of 92 kDa ge latinase and dipeptydyl-pe ptidase-IV in the se processes in the course of MS. 8,9 The results of our study suggest than APN may also partic ipate in it.
We have show n the pre senc e of acivated PBMC in the pe rip heral blood of MS patients. In the c ourse of acute ex acerbation and in CP-MS, these ce lls show a pote ntially greate r ability to cross the BBB. The data obtained may suggest that PBMC might play an active role in BBB disruption in the course of MS.