Impact of type-I-interferon on monocyte subsets and their differentiation to dendritic cells

Journal of Neuroimmunology 146 (2004) 176 – 188 www.elsevier.com/locate/jneuroim

Impact of type-I-interferon on monocyte subsets and their differentiation to dendritic cells An in vivo and ex vivo study in multiple sclerosis patients treated with interferon-beta F. Then Bergh a,b,*, Farshid Dayyani a, Loems Ziegler-Heitbrock a,c a Institute of Immunology, Ludwig-Maximilians-Universita¨t, Munich, Germany Institute of Clinical Neuroimmunology, Ludwig-Maximilians-Universita¨t, Munich, Germany c Clinical Cooperation Group ‘‘Inflammation of the Lung’’, Institute of Inhalationbiology, GSF - National Research Center for Environment and Health, Asklepios-Klinik, Gauting, Germany b

Received 5 September 2003; received in revised form 8 October 2003; accepted 8 October 2003

Abstract In addition to CD14++ ‘‘classical’’ monocytes, human peripheral blood contains CD14 + CD16+ ‘‘pro-inflammatory’’ monocytes, which may be influenced by IFNb treatment. By fluorescence activated cell sorting (FACS) analysis, 94 multiple sclerosis (MS) patients revealed normal absolute and relative numbers of both monocyte populations (71 untreated, 23 IFNb-treated). In IFNb-treated patients, CD14 + CD16+ monocytes consistently expressed higher CD14, confirmed in 16 patients analyzed longitudinally. Ex vivo, CD1a + CD14+ dendritic cells (DC) were efficiently differentiated from peripheral blood cells from controls and untreated patients, but at considerably reduced efficiency in IFNb-treated patients. Addition of IFNb to the medium further reduced the induction of CD1a + CD14+ cells. IFNb induces a novel immunophenotypic shift in pro-inflammatory monocytes, which appears to be related to reduced formation of dendritic cell precursors. D 2003 Elsevier B.V. All rights reserved. Keywords: Dendritic cell; Interferon-beta; Multiple sclerosis; Monocyte

1. Introduction Monocytes constitute between 3% and 7% of peripheral blood leukocytes. They derive from progenitors in the bone marrow, circulate in the blood as monocytes, and in tissue can differentiate into either macrophages or dendritic cells. The classical immunophenotypic marker for monocytes is CD14, the lipopolysaccharide receptor. Within this population, about 10% co-express CD16 (the low-affinity Fcgamma receptor III). Since the first description of this CD14 + CD16+ monocyte subpopulation, we have been able

Abbreviations: DC, dendritic cell; FACS, fluorescence activated cell sorting; IFN, interferon; MS, multiple sclerosis. * Corresponding author. Current address: Klinik und Poliklinik fu¨r Neurologie, Universita¨tsklinikum Leipzig, Liebigstr. 22a, D-04103 Leipzig, Germany. Tel.: +49-341-97-24255; fax: +49-341-97-24239. E-mail address: [email protected] (F. Then Bergh). 0165-5728/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.neuroim.2003.10.037

to show that they express (1) higher levels of MHC class II (Ziegler-Heitbrock et al., 1993), (2) little or no IL-10 mRNA in response to LPS-stimulation (Frankenberger et al., 1996), but (3) comparable levels of induced TNF-alpha mRNA (Frankenberger et al., 1996) and higher levels of TNF-alpha protein (Belge et al., 2002), compared to the ‘‘classical’’ (CD14 + CD16 ) monocytes. We have therefore termed them ‘‘pro-inflammatory monocytes.’’ Consistent with this notion, an expansion of the CD14 + CD16+ population has been found in septicemia (Fingerle et al., 1993), erysipelas (Horelt et al., 2002), AIDS and other infectious or inflammatory disorders (Ziegler-Heitbrock, 1996). Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). There is ample evidence to support an autoimmune pathogenesis of MS, crucially dependant on autoreactive T lymphocytes (Hohlfeld et al., 1995). Despite some variation among patients (Bru¨ck et al., 1995; Lucchinetti et al., 2000), the inflamma-

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tory lesions in the CNS white matter commonly contain considerable numbers of macrophages. While there is currently no cure for MS, acute exacerbations can be effectively treated with a pulse of high-dose glucocorticoids (Miller et al., 2000), and chronic interferon beta (IFNb) treatment reduces relapse frequency and disease progression (Goodin et al., 2002). We have previously shown that intravenous glucocorticoids selectively deplete the pro-inflammatory monocytes from the peripheral blood of MS patients (Fingerle-Rowson et al., 1998), which may contribute to the drug’s rapid anti-inflammatory effect. Here, we examined if the established long-term treatment with IFNb also has an effect on monocyte subsets. The CD14 + CD16+ subset of peripheral blood monocytes is also thought to be a transitional stage in the development of monocytes to either macrophages or dendritic cells (Ziegler-Heitbrock et al., 1993; Siedlar et al., 2000; de Baey et al., 2001). Within this population, those cells with low levels of CD14 appear to develop into dendritic cells (Siedlar et al., 2000; de Baey et al., 2001). We demonstrated in MS patients that IFNb will shift the CD14 + CD16+ towards higher expression of CD14. This may skew the developmental potential of monocytes in favour of macrophages, at the expense of dendritic cells. In fact, several in vitro studies suggest that IFNb inhibits development and function of dendritic cells (Bartholome et al., 1999b; McRae et al., 2000b; Duddy et al., 2001; Wiesemann et al., 2002), but these observations have not been confirmed in vivo. We therefore studied the effect of IFNb on ex vivo generation of dendritic cells and suggest that IFNb treatment inhibits the formation of dendritic cell precursors from monocytes.

2. Subjects and methods 2.1. Overall design Patients with clinically definite multiple sclerosis (Poser et al., 1983) of at least 18 years of age, who presented to the outpatient clinic of the Institut fu¨r Klinische Neuroimmunologie, were included in an open study. Patients were required to be free of corticosteroid medication for at least 2 months before any blood sampling. We excluded patients with other inflammatory or infectious diseases, any severe general medical illness or abnormal results of a routine laboratory investigation (blood chemistry, complete blood count, CRP, thyroid function tests, anti-nuclear antibodies, urinalysis). All patients gave informed consent. First, peripheral blood monocyte populations were analyzed in a cross-sectional study in 94 MS patients with different clinical course (relapsing-remitting, RR; secondary-progressive, SP; primary-progressive, PP). Seventy-one patients without immunomodulatory treatment were studied and 23 patients who were using one of the three approved preparations of interferon beta (IFNb 1a: RebifR, Serono

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Deutschland, Unterschleissheim or AvonexR, Biogen, Ismaning; IFNb 1b: BetaferonR, Schering, Berlin, all in Germany). To be included, treated patients had to be on the same treatment regimen for at least 6 months. This crosssectional study was performed from a single sample of venous blood. The results of the cross-sectional analysis were confirmed in a longitudinal study in 16 patients who initiated treatment with interferon-beta, based on clinical indication. This part of the study comprised blood samples taken before the first interferon injection, after 1 month and after 4 to 6 months of treatment. In order to minimize possible acute effects of interferon-beta, all samples were taken in the morning before the next scheduled injection, which was routinely selfadministered in the evening. The potential to differentiate monocytes into dendritic cells was then studied in four healthy volunteers, eight untreated and eight IFNb-treated MS patients, involving a single blood sample. See Section 3 and tables for demographic and clinical characteristics of the subjects. 2.2. Analysis of peripheral blood leukocytes Two millilitres of venous blood was drawn into EDTA containing syringes. A complete blood count was performed immediately, the remaining sample was stored at 4 jC and processed within 6 h. For flow cytometric analysis of monocyte subsets, 200 Al of peripheral blood was subjected to erythrocyte lysis (ammonium chloride), the leukocytes washed once and resuspended in 50-Al PBS/2%FCS. The cells were incubated for 20 min on ice with FITC-conjugated anti-human-CD14 (clone 322A-1 [My4], Beckman CoulterImmunotech, Krefeld, Germany), PE-conjugated anti-human-CD16 (clone B73.1, BD Pharmingen, Heidelberg, Germany) and PC5-conjugated anti-human-HLA-DR (clone Immu-357, Beckman Coulter-Immunotech). Control samples were prepared by incubation with the respective, fluorochrome-labelled isotypic control antibodies from the same manufacturers. After washing twice in PBS/2%FCS, cells were resuspended in PBS/2%FCS/0.1% sodium azide and analyzed within 1 h on a FACScan (BD Pharmingen). Using CellQuest software (BD Pharmingen), the FSC/SSC scatter plot was used to define a monocyte gate, within which the relative expression of CD14 and CD16 defined the two major monocyte subsets (Fig. 1). At least 10,000 monocytes (as defined by scatter gate and CD14/CD16 expression) were acquired. 2.3. Cell culture Four millilitres of venous blood was drawn into heparincoated syringes, stored at 4 jC and processed within 2 h. The blood was mixed with 10-ml culture medium (RPMI1640, Life Technologies, Grand Island, NJ, USA, supplemented with 10% fetal calf serum, L-glutamine and

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penicillin/streptomycin), washed twice in medium and the cells resuspended in a final volume of 4 ml medium. One millilitre each was incubated in 15-ml polypropylene tubes

at 37 jC and 5% CO2 for 24 h (a) without cytokines, (b) with granulocyte-monocyte-colony stimulating factor (GMCSF, 10 ng/ml, Essex Pharma, Mu¨nchen, Germany), inter-

Fig. 1.

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Table 1 Cross-sectional analysis of the immunophenotype of blood monocytes Group

Age Duration of WBC treatment (n/Al) (months)

Granulo Lympho Mono CD14 ++ CD14 + CD16 + (%) (%) (%) n/Al CD14 CD16 HLA-DR n/Al CD14 CD16 HLA-DR MFI MFI sMed MFI MFI sMed

MS (n = 71)

38.3 10.3 1.2 34.4 9.1 1.9 33.7 7.3 1.1 31.8 8.1 1.9 45.3 9.5 2.5 43.6 6.1 2.7 46.5 11.3 3.3

66.2 7.5 1.0 60.1* 9.5 2.1 66.2 8.2 1.4 58.8* 9.2 2.2 64.0 6.6 1.8 68.0 8.7 5.0 69.7 4.8 1.6

Mean S.D. S.E.M. MS-IFN (n = 23) Mean S.D. S.E.M. RR (n = 44) Mean S.D. S.E.M. RR-IFN (n = 18) Mean S.D. S.E.M. SP (n = 15) Mean S.D. S.E.M. SP-IFN (n = 5) Mean S.D. S.E.M. PP (n = 12) Mean S.D. S.E.M.

15.4 11.8 2.5

16.7 12.7 3.0

10.4 7.0 3.1

7340 1790 226 5318*** 1582 337 7225 1496 237 5172*** 1377 325 7429 1751 468 5975*** 2464 1232 7711 2953 984

24.5 6.7 0.9 29.6* 8.4 1.8 24.8 7.3 1.2 30.3* 8.3 1.9 25.9 5.7 1.6 25.0 9.9 5.7 21.4 4.1 1.4

7.1 2.2 0.3 8.9* 4.0 0.9 6.7 2.5 0.4 9.4* 4.1 1.0 8.0 1.7 0.5 5.7 1.2 0.7 7.1 1.3 0.4

235.4 121.0 15.1 244.1 140.5 30.7 240.1 115.3 18.0 244.7 104.4 25.3 261.5 145.7 38.9 241.7 271.1 135.6 173.4 93.4 31.1

346.2 158.5 19.7 397.6 153.3 32.7 358.6 151.0 24.5 397.4 157.0 37.0 340.6 174.1 45.0 398.3 157.5 78.8 313.8 170.9 49.3

4.3 2.3 0.3 4.7 2.6 0.6 3.9 1.2 0.2 4.8 2.6 0.6 4.4 3.0 0.8 4.2 3.0 1.5 5.1 3.7 1.1

40.7 27.9 4.5 32.2 36.7 10.6 46.4 31.6 6.7 33.8 42.9 14.3 35.3 25.4 9.0 27.3 4.7 2.7 30.7 15.2 5.4

30.0 63.3 427.1 161.7 18.0 30.9 271.0 138.7 2.3 3.8 33.6 22.8 39.8 120.0*** 377.1 169.7 23.8 34.0 197.7 235.9 5.2 7.2 42.1 68.1 30.3 66.3 449.6 180.4 19.8 28.9 232.2 148.5 3.1 4.7 37.7 31.7 41.2 120.4*** 410.2 189.0 26.3 31.2 203.0 273.3 6.4 7.4 47.9 91.1 33.4 58.0 379.5 141.7 17.3 37.7 269.6 152.6 4.6 9.7 69.6 53.9 33.9 117.8*** 228.3 111.5 5.9 50.5 62.4 24.3 3.0 25.3 31.2 14.0 23.6 60.3 415.5 126.1 7.2 29.3 385.2 88.0 2.4 8.5 111.2 33.3

*p < 0.05, **p < 0.01, ***p < 0.001 for comparison of IFN-beta-treated vs. respective untreated group (ANOVA and post-hoc comparisons).

leukin-13 (IL-13, 100 ng/ml, kind gift from Dr. Bartholeyns, IDM, Paris, France) and tumor-necrosis-factor (TNF, 100 U/ml, BASF-Knoll, Ludwigshafen, Germany), (c) with GM-CSF, IL-13, TNF and interferon beta-1a (IFN-beta, 50 ng/ml, RebifR lot no. 84517, Serono), or (d) with IFN-beta alone. After incubation, cells were harvested by centrifugation, resuspended, stained and analyzed by flow cytometry as described above. The antibodies used were FITC-conjugated anti-human-CD14 (clone 322A-1 [My4]), PE-conjugated anti-human-CD1a (clone HI149, BD Pharmingen), PE-conjugated anti-human CD83 (clone HB15e, BD Pharmingen) and PC5-conjugated anti-human-CD16 (clone 3G8, CoulterImmunotech). In order to minimize day-to-day variations, cytokines were stored in aliquots at 80 jC and thawed immediately before addition to the cell suspension. In addition, cell culture experiments were always performed in parallel, using blood samples obtained on the same morning from a healthy

control or untreated patient as well as an interferon-treated patient. 2.4. Statistical analysis Relative numbers, mean and median fluorescence intensities were recorded for each subset of cells, as defined by regions in two-colour scatter plots. Absolute cell counts were calculated based on the complete leukocyte count. The level of HLA-DR expression was determined from frequency histograms, as the difference of the median channels of the specific antibody sample vs. the isotypic control (specific median, sMed). Cross-sectional differences between groups of patients or between controls and patients were statistically analyzed by analysis of variance (ANOVA), with weighting where appropriate. Longitudinal effects of interferon-beta on blood monocytes were analyzed by multivariate F-tests with ‘‘time’’ as within-subject-factor (with three levels:

Fig. 1. (A) Flow cytometric analysis of peripheral blood monocyte subsets. (Panel A) Shown is the definition of the monocyte gate according to forward (FSC) and sideward (SSC) light scatter. (Panel B) Within the light scatter gate, monocytes are separated into the ‘‘classical’’ CD14++ phenotype and an additional CD14 + CD16+ population. CD14 CD16+ cells are natural killer (NK) cells. (Panel C) Representative analysis from a multiple sclerosis patient treated with IFNb. The CD14 + CD16+ cells express higher levels of CD14, with a clear gap to the NK cells. (Panels D and E) The expression of HLA-DR on both monocyte subsets is not significantly different between untreated MS patients (D) and patients on long-term IFNb treatment (E). (B) Increased CD14 expression on CD14 + CD16+ monocytes with IFN-beta treatment. Boxplot of CD14 mean fluorescence intensity (CD14 MFI) on the CD14 + CD16+ monocyte subset (+ marks indicate the 95% confidence interval) in different groups of MS patients. Patients treated with IFNb had higher CD14 expression than untreated patients (MS-IFN vs. MS, p < 0.001). Differences were equally significant when patients were separated according to clinical course (RR, relapsing – remitting; SP, secondary-progressive; PP, primary-progressive).

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Fig. 2. Time course of antigen expression on monocyte subsets. (A) In this representative patient with SP-MS, initiation of IFNb therapy leads to higher CD14 expression on CD14 + CD16+ monocytes within 1 month (note the shift to the right). At the same time, HLA-DR expression increases, particularly on CD14 + CD16+ cells (solid lines: HLA-DR, broken lines: isotypic control). (B) Time course of CD14 and HLA-DR expression on both monocyte subsets. Solid line: All MS patients; dashed lines, filled circles: RR-MS; dashed line, open circles: SP-MS.

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baseline, 1 month and 3 months treatment duration). ‘‘Course’’ (with two levels for relapsing-remitting or secondary-progressive) and ‘‘treatment’’ (three levels for the three different preparations of IFNb) were entered as

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between-subjects-factors; age and gender were analyzed as covariates. The effects of IFNb on in vitro generation of dendritic cells was evaluated by multivariate F-tests, with ‘‘interferon’’ as within-subjects factor (two levels:

Table 2 Longitudinal analysis of peripheral blood monocytes in MS patients starting IFN-beta therapy All patients (n = 16)

S.D.

S.E.M.

Mean Age EDSS Baseline WBC Granulo Lympho Mono CD14 ++

CD14 + 16 +

33.5 2.9 n/Al % % % n/Al CD14 MFI CD16 MFI HLA-DR, sMed n/Al CD14 MFI CD16 MFI HLA-DR, sMed

1 month IFN-beta preparation IFNb 1a (Rebif22R)/ IFNb 1a (Avonexk)/ IFNb 1b (BetaferonR) WBC n/Al Granulo % Lympho % Mono % CD14 ++ n/Al CD14 MFI CD16 MFI HLA-DR, sMed CD14 + 16 + n/Al CD14 MFI CD16 MFI HLA-DR, sMed 3 months WBC n/Al Granulo % Lympho % Mono % CD14 ++ n/Al CD14 MFI CD16 MFI HLA-DR, sMed CD14 + 16 + n/Al CD14 MFI CD16 MFI HLA-DR, sMed Statistics (Variables not listed here: n.s.) WBC n/Al Mono % CD14 + + HLA-DR, sMed CD14 + 16 + n/Al CD14 MFI HLA-DR, sMed

7113 64.8 25.6 7.4 248.0 320.9 3.6 38.9 32.8 54.3 388.2 168.5

S.D.

S.E.M.

Mean 8.6 1.8

2.1 0.4

1264 6.6 5.9 2.0 127.1 161.3 1.4 25.9 15.1 18.2 234.4 162.6

316 1.7 1.5 0.5 31.8 40.3 0.3 6.9 3.8 4.5 58.6 43.4

8/3/5

5613*** 60.1 28.4 9.3** 230.2 352.9 6.0 72.9** 40.5 121.3*** 355.5 388.8**

RR (n = 12)

31.3 2.0 6742 64.2 26.5 6.9 215.4 302.0 3.8 42.2 30.6 56.9 458.1 195.3

7.0 0.6

2.0 0.2

951 275 7.4 2.1 6.5 1.9 2.0 0.6 60.2 17.4 155.8 45.0 1.5 0.4 27.8 8.4 15.9 4.6 18.0 5.2 227.9 65.8 174.9 52.7

8/3/1

1526 7.3 7.2 2.8 89.8 90.0 9.4 61.3 19.1 28.4 233.2 324.1

381 1.8 1.8 0.7 22.4 22.5 2.3 15.8 4.8 7.1 58.3 83.7

5213 1367 342 57.9 8.4 2.1 30.0 6.4 1.6 10.1 3.0 0.7 267.9 128.7 32.2 300.3 63.6 15.9 3.8 1.3 0.3 96.8 69.2 17.3 66.6* 39.2 9.8 95.3 33.6 8.4 453.9 338.0 84.5 401.6 258.6 64.7 p (Multivariate F-test: p < 0.05 (Sequential effect of time) contrasts) < 0.001 Base vs. 1 mo 0.003 Base vs. 1 mo 0.007 Base vs. 1 mo 0.018 1 mo vs. 3 mo < 0.001 Base vs. 1 mo 0.004 Base vs. 1 mo

5558 60.3 28.8 8.6 227.0 371.4 7.0 91.9 41.5 127.6 432.1 492.7

SP (n = 4)

S.D.

S.E.M.

Mean 40.3 5.6 8225 66.5 23.0 8.8 345.6 377.5 2.9 27.0 39.3 46.5 178.5 70.3

10.4 0.9

5.2 0.4

1571 786 3.7 1.8 2.4 1.2 1.3 0.6 224.7 112.4 188.0 94.0 0.5 0.2 14.6 8.4 11.7 5.9 18.8 9.4 78.8 39.4 20.1 11.6

0/0/4

1543 445 7.9 2.3 6.8 2.0 2.4 0.7 70.1 20.2 89.9 26.0 10.8 3.1 61.3 18.5 20.3 5.8 28.6 8.3 218.2 63.0 320.0 96.5

5775 59.5 27.5 11.3 239.9 297.3 3.1 20.6 37.4 102.3 125.8 103.1

1692 846 5.8 2.9 9.3 4.7 3.2 1.6 148.7 74.4 73.3 36.7 0.8 0.4 6.6 3.3 17.3 8.7 19.6 9.8 59.6 29.8 19.4 9.7

5033 1343 388 5750 1488 57.6 8.6 2.5 58.8 9.0 30.8 6.6 1.9 27.8 6.4 9.4 2.7 0.8 12.0 3.2 224.7 92.1 26.6 397.6 147.7 286.5 63.8 18.4 341.8 47.4 4.0 1.5 0.4 3.3 0.3 113.0 72.5 20.9 48.1 21.1 60.1 37.7 10.9 86.0 42.5 84.6 30.7 8.9 127.5 18.4 548.5 338.0 97.6 170.3 97.2 461.0 270.1 78.0 223.6 102.8 p (Multivariate F-test: effect of time by course)

0.047

*p < 0.05, **p < 0.01, ***p < 0.001 for time effect (multivariate F-test and significant contrast vs. the previous time point).

744 4.5 3.2 1.6 73.9 23.7 0.2 10.6 21.3 9.2 48.6 51.4

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absent and present in culture medium), and ‘‘group’’ as between-subjects factor (three levels: control, untreated and IFNb-treated patient). In addition, paired sample t-tests were performed. P < 0.05 was accepted as the level of statistical significance. For multiple comparisons, ANOVA or multivariate tests were followed by localization of group differences, using post-hoc pairwise comparisons and adjustments according to Bonferroni’s procedure. SPSS 11.0 for Windows was used for all statistical procedures. Data are given as mean F S.D. (standard deviation) and S.E.M. (standard error of the mean).

in 94 MS patients (Table 1). The absolute and relative numbers were in the range of previously published values (Ziegler-Heitbrock, 1996). In untreated patients, no significant differences were found according to the clinical course of the disease (relapsing-remitting (RR), secondary-progressive (SP) or primary-progressive (PP)), although patients with the primary progressive form had a higher proportion of CD14 + CD16+ cells among all monocytes (see Table 1).

3. Results

When we compared monocyte subsets in patients who were being treated with IFNb to those in untreated patients, the CD14 + CD16+ monocytes in treated patients consistently expressed higher levels of CD14 (Fig. 1A, panels B vs. C, Fig. 1B and Table 1). This difference was highly significant ( p < 0.001, ANOVA), irrespective if IFNb-treated patients were analyzed as a group or separated according to the

3.1. MS patients have normal absolute and relative numbers of peripheral blood monocytes In a cross-sectional study, we first analyzed absolute and relative numbers of peripheral blood monocyte subsets

3.2. In IFN-beta treated patients, CD14+CD16+ monocytes express higher levels of CD14

Fig. 3. Induction of dendritic cell markers in vitro. Culture of whole peripheral blood in the presence of GM-CSF, IL-13 and TNF results in the generation of cells expressing the dendritic cells markers CD1a+ (top) and CD83+ (bottom) within 24 h. Most CD1a+ cells were CD14+, while CD83+ cells were CD14 and by back-gating located to the lymphocyte light scatter region.

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particular IFNb preparation. Also, the difference remained highly significant when treated and untreated patients of the same clinical course were compared (relapsing-remitting or secondary progressive; Fig. 1B and Table 1). The increase in mean CD14 expression was at the expense of those

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CD14 + CD16+ cells with very low CD14; these latter cells were essentially absent (Fig. 1A, panel C). The ‘‘classical’’ CD14+ monocytes showed little difference between untreated and IFNb treated patients. Complete and differential leukocyte count, provided by routine automated methods,

Fig. 4. Effect of IFNb on CD1a + CD14+ dendritic cells in vitro. (Panel A, B) In vitro generated CD1a+ cells can be defined as monocyte-derived dendritic cells by co-expression of CD14. The percentage of CD1a + CD14+ cells among all CD14+ cells is given for each panel. CD1a+ cells equally develop in the CD14low and CD14high population. In this representative example, CD1a+ cells accounted for 11.9% of CD14low and 12.9% of CD14high cells. (Panel C) IFNb 1a reduces the percentage of CD1a+ dendritic cells among CD14+ cells. The reduction was seen both in CD14low (8.5% CD1a+) and CD14high cells (4.4% CD1a+). (Panels D, E) Similar results were obtained from untreated MS patients. Again, IFNb in vitro reduced the proportion of CD1a+ in CD14low (in this patient: 7.0% without IFNb vs. 0.90% with IFNb) as well as in CD14high cells (6.0% vs. 0.46%). (Panels F, G) Chronic IFNb therapy results in significantly diminished efficiency to generate CD14 + CD1a+ dendritic cells even without addition of IFNb to the culture medium. Here, only 2.84% of CD14low monocytes developed into CD1a+ cells, and 2.25% of the CD14high population. Additional presence of IFNb in vitro virtually abolished the formation of CD1a+ cells among either CD14low (0.23% CD1a+) or CD14high cells (0.06% CD1a+).

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showed changes as previously described during IFNb treatment, including a lower total leukocyte number and relative lymphopenia. 3.3. The differences in CD14 expression develop within 1 month To verify that the differences in antigen expression were indeed due to IFNb treatment, we then longitudinally studied 16 patients who started therapy. Fig. 2A shows a representative example of the time course: in the CD14 + CD16+ monocytes, a shift toward higher expression of CD14 and HLA-DR is detected within 1 month and persists at 3 months. This observation was made in all 16 patients, to different degrees (Fig. 2B). The differences were highly significant in multivariate F-tests (CD14: p < 0.001; HLA-DR: p = 0.004); tests with contrasts confirmed that the changes occurred between baseline and 1 month of treatment (see Table 2). The absolute count of CD14 + CD16+ cells increased approximately twofold, mainly between month 1 and month 3. Although the time course of CD14 expression on CD14 + CD16+ cells was slightly different between patients with relapsing –remitting and secondary-progressive MS, the clinical course generally had no effect on the other variables studied (see Table 2). The CD14+ ‘‘classical’’ monocytes did not change in their levels of CD14 or CD16 expression, but showed slightly increased HLA-DR expression ( p = 0.007, multivariate F-test). Table 2 lists the data for the entire group. 3.4. Generation of CD1a-positive dendritic cells from peripheral blood is inhibited by IFN-beta in vitro We developed a protocol to generate CD1a+ dendritic cells from whole peripheral blood within 24 h of culture (see Section 2). CD1a+ cells generated in this way were primarily CD14+, i.e., a phenotype characteristic of monocyte-derived dendritic cells. Cells expressing CD83, on the other hand, were almost exclusively CD14 , and located in the lymphocyte light scatter region (see Fig. 3). Addition of TNF was necessary to obtain sufficient expression of CD1a within this short culture period. Extending the culture period up to 7 days (with replenishment of medium and cytokines every other day) resulted in higher absolute numbers, but comparable proportions of CD1a+ and CD83+ cells as the short-term culture (not shown). However, the light scatter characteristics and expression of monocyte markers were increasingly altered, making it more difficult to define their derivation from monocytes. We therefore chose the 24-h protocol with addition of TNF for all further experiments, and focused on CD1a expression as marker for monocyte-derived dendritic cells. In healthy controls, no CD1a+ cells were detected in freshly obtained peripheral blood; culture without cytokines did not result in any detectable CD1a+ cells, either (data not shown). Addition of GM-CSF, IL-13 and TNF-alpha

Table 3 Percentage of CD1a+ dendritic cells among CD14+ cells, after 24-h incubation with GM-CSF, IL-13 and TNF, in the absence ( IFN) or presence ( + IFN) of interferon-beta 1a Group

Controls

MS

MS-IFN

p

IFN

n=4 Mean 9.84 S.E.M. 3.20 n=8 Mean 8.26 S.E.M. 1.31 n=8 Mean 4.31** S.E.M. 1.13 Multivariate F-test < 0.001 (effect of group by IFN) ANOVA (effect of group) 0.02

+ IFN

p, Paired t-test (effect of IFN in vitro) < 0.002

2.37 0.68 2.59 1.00 1.39** 0.20

0.049

** p < = 0.003 vs. controls in post-hoc comparisons with Bonferroni correction.

resulted in the generation of a new population of CD1a+ cells in the CD14+ compartment (see Fig. 4B). The induction of CD1a was seen in both the CD14low and the CD14high cells, but with the low number of events, we performed a combined analysis for this study. These dendritic cells (DC) comprised a mean of 9.8% of the CD14+ cells (see Table 3). CD1a+ cells could be generated from blood samples from healthy controls and untreated MS patients with comparable efficiency (Fig. 4, compare panel D to panel B; Table 3). IFNb significantly inhibited the formation of monocytederived CD1a+ DC. As shown in Fig. 4C for a representative example and summarized in Table 3, only few CD14 + CD1a+ cells were generated in the presence of IFNb ( p < 0.001, paired samples t-test). Culture with IFNb alone (without GM-CSF, IL-13 and TNF) resulted in a slight up-regulation of CD14 on classical monocytes; no CD1a+ cells were generated (data not shown). 3.5. The efficiency of generating CD1a+ dendritic cells from peripheral blood is diminished in IFN-beta-treated patients Compared to healthy controls and untreated patients, dendritic cells could be generated with significantly reduced efficiency in blood samples from IFNb-treated patients upon culture with GM-CSF, IL-13 and TNF (Fig. 4F). On average, 4.3% of CD14+ cells expressed CD1a, as opposed to 9.8% in controls and 8.3% in untreated patients ( p = 0.02, ANOVA). Addition of IFNb to the medium virtually abolished the differentiation of CD14+ cells to CD1a+ dendritic cells (Fig. 4G). This was again highly significant when analyzed with a paired samples t-test for the IFN-treated patients separately ( p = 0.002). As a global statistical analysis, a multivariate F-test was equally significant ( p < 0.001, comparing the three donor groups with respect to the

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percentage of CD1a + CD14+ cells, according to IFN addition to the culture medium; see Section 2). Table 3 summarizes the results of the in vitro DC generation experiments.

4. Discussion In this open study in multiple sclerosis patients, we report that treatment with interferon-beta is associated with a shift toward higher CD14 expression on the pro-inflammatory subset of peripheral blood monocytes. Further, we demonstrate that IFNb appears to inhibit the formation of monocyte-derived progenitors for dendritic cells in vivo, and decreases their further differentiation in vitro. 4.1. Monocytes in multiple sclerosis The presence of macrophages in demyelinating lesions has been demonstrated in patients with multiple sclerosis (Bru¨ck et al., 1995) as well as in experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS (Rinner et al., 1995). Formal proof for the derivation of these cells from bone marrow has been obtained in the animal model, by induction of EAE in bone marrow-chimeric or transgenic animals (Hickey and Kimura, 1988; Flu¨gel et al., 2001). An analogous situation is commonly assumed, but has yet to be demonstrated, in MS. With respect to peripheral blood monocytes, the presumed intermediates between bone marrow and tissue macrophages, numerous studies have addressed various properties of mononuclear cell preparations in MS, but analyses of monocyte populations are limited. We found normal absolute and relative numbers of the two main subsets and levels of HLADR expression within the range determined previously (Ziegler-Heitbrock et al., 1993; Fingerle et al., 1993; Horelt et al., 2002). We could not detect significant relationships to clinical characteristics, although our patient sample would probably be large enough to define such relationships. The data obtained here agree well with our earlier study in a smaller patient sample (Fingerle-Rowson et al., 1998). Another recent study (Kouwenhoven et al., 2001) examined monocytes obtained by density gradient separation and found that untreated MS patients (as well as control patients with other neurological diseases) had normal percentages of CD16+ and CD64+ cells within that preparation. Thus, although some degree of systemic immune dysregulation has been described in MS, this obviously does not extend to monocyte subpopulations in the same manner as in infectious diseases. 4.2. Interferon-beta and the time course of HLA-DR expression The therapeutic benefit of interferon-beta in MS has been proven in several large clinical trials. While it was initially studied under the hypothesis of its anti-viral activity against a

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presumed infectious cause of MS, interferon-beta is believed to act through several different mechanisms (Yong et al., 1998). One prominent model is based on the observation that IFNb inhibits the IFN-gamma induced up-regulation of MHC class II molecules on the surface of macrophages (Ling et al., 1985) and glial cells (Barna et al., 1989; Jiang et al., 1995; Hall et al., 1997; Ransohoff et al., 1991; Satoh et al., 1995) and therefore diminishes antigen presentation inside the CNS. In apparent contrast to this, IFNb has been shown to increase HLA-DR expression on peripheral blood monocytes over short (Spear et al., 1987; Soilu-Hanninen et al., 1995) and longer time periods of up to 1 year (Gelati et al., 1999). Those studies relied on light scatter characteristics to define the monocyte population gate. From our longitudinal data over the first 3 months of therapy, we can further define that HLA-DR expression increases mainly on the CD14 + CD16+ subset, but also on the CD14 + CD16 monocytes. However, in the cross-sectional part of our study, HLA-DR was not increased in patients treated for at least 6 months, vs. untreated patients. We assume that either HLADR gradually decreases over time or patients treated for several years particularly contributed to this effect in our cross-sectional study. Alternatively, it may indicate a differential decrease in immunological effects among patients. Both would be consistent with the wider range of HLA-DR expression that we found in the treated patients. 4.3. A novel phenotypic shift in the pro-inflammatory monocytes Treatment with IFNb was consistently associated with a phenotypic change of the CD14 + CD16+ monocyte subpopulation that so far has not been reported: higher expression levels of CD14, in other words, a shift from CD14dimCD16bright towards CD14brightCD16bright. This was apparent from the cross-sectional part of the study, and confirmed in the longitudinal study of an additional group of patients starting IFNb treatment. The significance of this change is not immediately clear, but it may be related to the differentiation potential of blood monocytes. Monocytes develop into either macrophages or dendritic cells. While both of these cell types present antigen and are thus critically involved in immune activation, it is incompletely understood what determines the direction of monocyte differentiation, as are intermediate stages of these processes. Several recent studies suggest that the CD14 + CD16+ monocytes are intermediates in the differentiation of dendritic cells. In peripheral blood, dendritic cell precursors were suggested to be included in the CD64 + CD16+ subpopulation of monocytes (Grage-Griebenow et al., 2001). In a model of spontaneous development of dendritic cell precursors (Lin HLA-DR+) from PBMC, depletion of CD16+ cells delayed, and depletion of CD14+ cells abolished the formation of Lin HLA-DR+ cells (Ho et al., 2002). Sorted CD14 + CD16 as well as CD14 + CD16+ monocytes can be differentiated to dendritic cells, but those

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derived from CD16+ cells display higher CD86 expression and lymphocyte activation activity (Sanchez-Torres et al., 2001). More specifically, those monocytes with high expression of CD16, but low levels of CD14, appear to be precursors for dendritic cells, as they express the M-DC8 antigen (Siedlar et al., 2000; de Baey et al., 2001; Schakel et al., 1998). Since we found that IFNb treatment was associated with a decrease in precisely this group of cells (CD14dimCD16bright), we next studied the effect of IFNb on dendritic cell differentiation in vitro and ex vivo. 4.4. A simplified protocol to generate dendritic cells in vitro In recent years, several protocols have been described to generate dendritic cells from human bone marrow, cord blood and peripheral blood monocytes (reviewed by Ardavı´n et al., 2001). For the latter, the procedures commonly include isolation of monocytes by density gradient centrifugation and culture for several days in the presence of granulocyte-macrophage colony stimulating factor (GMCSF) and interleukin-4 (IL-4). IL-13 has been shown to be equally efficient as IL-4 (Piemonti et al., 1995). We adapted this general strategy for two reasons. First, we avoided density gradient centrifugation because it may cause adhesion and activation of monocytes and might alter the relative yield of different cell populations. Using whole blood also retains the general cellular environment present in the blood, which presumably reflects a more physiological situation. In this respect, it was recently shown that human peripheral blood mononuclear cells (PBMC) can sustain and generate functional dendritic cell precursors (Lin HLA-DR+), without the need for exogenous cytokines (Ho et al., 2002). Second, we shortened the incubation period to 24 h, after which CD14 expression was retained and allowed us to identify monocyte-derived cells. Addition of TNF-alpha was necessary to achieve sufficient maturation into dendritic cells expressing CD1a from both CD14low and CD14high monocytes. The proportion of dendritic cells generated after this short incubation is considerably smaller than what has been described for longer culture (1 week or more; Sallusto and Lanzavecchia, 1994; Romani et al., 1996). However, the results were sufficiently consistent to detect differences among groups of donors, as well as the effect of an additional cytokine (IFNb, see below). 4.5. The impact of interferon-beta on dendritic cell differentiation in vitro Using our abbreviated protocol, we found that IFNb reduced the proportion of monocyte-derived dendritic cells (CD14 + CD1a+) in vitro. This is in agreement with earlier studies in which IFNb decreased the generation of dendritic cells from human monocytes (Bartholome et al., 1999a,b; McRae et al., 2000b; Duddy et al., 2001; Huang et al., 2001; Wiesemann et al., 2002). IFN-alpha, the other type-I inter-

feron, has similar effects (Wang et al., 1999; Paquette et al., 1998). This diminished yield of DC in the presence of type-IIFN has been suggested to be at least partly due to induction of apoptosis (Lehner et al., 2001; Wiesemann et al., 2002). In monocyte-derived DC, IFNb reduces the production of IL12p40 through interference with the CD40L/CD40 pathway (Bartholome et al., 1999b; McRae et al., 1998, 2000a). The expression of costimulatory molecules (CD80, CD86) was found either reduced (McRae et al., 2000b) or increased (Bartholome et al., 1999b; Hussien et al., 2001; Wiesemann et al., 2002) in the different studies. In contrast to these overall inhibitory effects, others found that type-I interferons accelerated the maturation of dendritic cells from CD34+ progenitors (Luft et al., 1998) or monocytes (Santini et al., 2000), based on the expression of surface antigens, activity of lymphocyte stimulation or/and activity in hu-SCID mouse models. Notably, the study by Luft et al. (1998) is the only one that employs a rigorous serum-free culture protocol, which may explain some of the differences to the other studies. It has been suggested (Huang et al., 2001; Buelens et al., 2002) that IFNb induces monocytes to develop into Th2supporting type 2, rather than into Th1-inducing type 1 dendritic cells, a concept which may be able to reconcile some of the abovementioned discrepancies. 4.6. The effect of interferon-beta on dendritic cell precursors in vivo Several in vitro studies have addressed the effects of IFNb on monocytes (see previous paragraph), and most found that it decreased the differentiation of dendritic cells from monocytes and/or their function. Our observation that the generation of CD1a + CD14+ cells was reduced in patients chronically treated with IFNb extends those findings to the in vivo situation. The present data suggest that IFNb in vivo inhibits formation of monocytic (CD14+) cells that act as precursors for dendritic cells when exposed to GM-CSF and IL-13 ex vivo. Alternatively, IFNb treatment may predispose monocytes to undergo apoptosis rather than develop into dendritic cells, similar to findings in vitro (Lehner et al., 2001; Wiesemann et al., 2002). In conclusion, we assume that IFNb in vivo interferes with development of dendritic cell precursors, reflected by an altered phenotype of the pro-inflammatory monocytes, and resulting in a reduced number of antigen-presenting dendritic cells. This may constitute an additional mechanism of action contributing to the beneficial effect of IFNb in multiple sclerosis.

Acknowledgements The authors would like to thank Dr. R. Hohlfeld for continued support at all phases of the study and for critically reviewing the manuscript. We would also like to thank Dr.

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A. Yassouridis for reviewing the statistical procedures. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG ZI 288/2) to LZH and an unrestricted research grant from Serono Deutschland GmbH to the Institute of Clinical Neuroimmunology.

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