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Cattedra di Virologia, Università degli Studi di Milano;
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Cattedra di Immunologia, Universita’ degli Studi di Milano, Padiglione L.I.T.A., (Laboratorio Interdisciplinare Tecnologie Avanzate), Ospedale L. Sacco;
Laboratorio di Biologia and
Unità Sclerosi Multipla, Don C. Gnocchi Foundation, IRCCS (Istituto di Ricerca e Cura a Carattere Scientifico); and
§
Istituto di Igiene e Medicina Preventiva, Università degli Studi di Milano,
¶
Associazione Ricerca Schizofrenia ARS, Milan, Italy; and
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DNAX Research Institute, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytokines have been involved in the pathogenesis of MS; thus, increased
levels of TNF-
and IFN
were detected in MS patients with acute
disease (9, 10, 11, 12), and the number of TNF- and IFN mRNA-expressing
lymphocytes was suggested to be different in MS patients during
different phases of disease activity (13, 14, 15). Additionally, it was
observed that myelin Ag-T lymphocytes produce high quantities of
IFN- upon restimulation with these Ags in vitro (16, 17, 18). Finally,
treatment of MS with IFN- exacerbates the disease (19, 20), whereas
therapeutic approaches based on the utilization of IFN- and IFN-ß,
which are known to be effective in a number of patients (21, 22),
decrease IFN- production by T lymphocytes, suggesting a direct
pathogenetic role for this cytokine.
Because different cytokines preferentially activate CMI or humoral
immunity (23, 24), we analyzed IFN-, IL-2, and IL-12 (type 1
cytokines) as well as IL-10 (type 2 cytokine) production by PBMC of MS
patients with acute (AMS) or stable (SMS) disease and compared the
results with those obtained in a group of healthy donors. To extend our
analysis, we also examined a family of phenotypic markers (SLAM and
CD7) (25, 26) recently suggested to be expressed by T lymphocytes that
preferentially produce type 1 cytokines upon restimulation in the same
patients and controls. Because B7-1 and B7-2 are suggested to be
differentially expressed in chronic immune responses (27, 28), we also
analyzed the expression of B7-1 and B7-2 on the surface of
macrophage/monocytes (CD14+). Finally, because 1) increased
susceptibility of immune cells to apoptosis is postulated as a
mechanism that is potentially protective against immune-mediated
destruction of the myelin sheath in MS (29); 2) the CD95/CD95
(Apo-1/Fas) ligand system is a key mediator of apoptosis (30); and 3)
soluble Apo-1 (sApo-1)/Fas was hypothesized to protect lymphocyte
against apoptosis (31), we measured the expression of Apo-1/Fas on
immune cells of MS patients and controls, and the serum concentration
of sApo-1/Fas in the same individuals.
Our results indicate that a profound and complex immune imbalance is present in MS. This imbalance is reflected in the impairment of cytokine production, in the overexpression of particular phenotypic populations (including Th1 markers expressing T lymphocytes), and in the dysregulation of surface Apo-1/Fas and its soluble ligand.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Forty-eight patients with MS characterized by clinical and laboratory criteria and followed by the Centro Sclerosi Multipla of the Don Gnocchi Foundation (Milan, Italy) were included in the study. These patients (30 women and 18 men; median age, 30.1; range, 21.3–40.4 yr) were affected by relapsing-remitting (RR) MS. RR MS had been clinically stable for at least 1 mo before the study period in 26 patients; these patients (median age, 31.4; range, 20.7–40.4 yr; 13 women and 13 men) were therefore classified as patients with SMS. The diagnosis of SMS was confirmed in these individuals by magnetic resonance imaging with gadolinium, which did not show areas of enhancement. The other 22 RR MS patients (median age, 30.8; range, 21.3–38.2 yr; 17 women and 5 men) presented with clinical re-exacerbation of the disease and were therefore classified as patients with AMS. AMS patients were visited and blood sampling was performed within 5 days of the onset of the acute episode and always before the onset of steroid-based therapy. Disease duration was comparable in the two groups (mean duration ± SD: SMS, 6.6 ± 6.6 yr; AMS, 7.0 ± 6.1 yr); none of the patients received immunosuppressive drugs in the year before the study period. Fifty-three healthy donors from the Transfusion Department of L. Sacco Hospital (Milan, Italy) were used as controls; median age (31.7; range, 22.8–40.7 yr) and sex (30 women and 23 men) were comparable between RR MS patients and controls. The blood samples were collected under protocols approved by both institutions.
Blood sample collection
Whole blood was collected by venipuncture in Vacutainer tubes containing EDTA (Becton Dickinson Co., Rutherford, NJ). PBMC were separated on lymphocyte separation medium (Organon Teknika Corp., Durham, NC) and washed twice in PBS, and the number of viable leukocytes was determined by trypan blue exclusion and a hemocytometer. The samples were kept at room temperature and used within 2 h.
In vitro Ag-stimulated IL-2 production
PBMC (3 x 105) were placed in flat-bottom wells of a microtiter culture plate (Costar, Cambridge, MA) in a final volume of 0.2 ml along with 1) no stimulation (medium background); 2) influenza virus vaccine prepared with a mixture of A/Taiwan, A/Shanghai, and B/Victoria (24 mg/l; final dilution, 1/1000); and 3) a pool of irradiated (50 Gy) allogeneic PBMC (1 x 105 cells/well) from two or more unrelated healthy control volunteers (HC). Three replicate cultures were performed for each stimulation. Pooled human plasma (1/20 final dilution) and the anti-IL-2R Ab, humanized monoclonal anti-Tac (Becton Dickinson; 1 µg/ml; to prevent uptake of the produced IL-2 and allow measurement of total IL-2 production during the 7 days of culture) were added to each well 1 h after sensitization of the PBMC. Culture supernatants were harvested after 7 days, and total IL-2 produced throughout the culture period was determined by testing each supernatant for the ability to stimulate the proliferation of an IL-2-dependent mouse continuous T lymphocyte line (CTLL). A standard curve (based on known quantities of human rIL-2) was ran in all the assays. Ag-stimulated IL-2 production was measured in a bioassay, whereas mitogen-stimulated IL-2 production was quantified with ELISA methods (see below). This choice was secondary to the fact that the sensitivity of bioassays is more adequate to measure relatively small quantities of IL-2, such as those produced upon antigenic stimulation. Five successive twofold dilutions in triplicate were set up to test the supernatants for the ability to stimulate the proliferation of 8 x 103 CTLL/well in 96-well microtiter plates. After 24 h, the CTLL cultures were pulsed with 1 µCi of [3H]thymidine and harvested 18 h later.
In vitro cytokine production
PBMCs resuspended at 3 x 106/ml in RPMI
1640 were either unstimulated or stimulated for 2 days with PHA (Life
Technologies, Grand Island, NY) diluted 1/100 (final concentration) at
37°C in a moist, 7% CO2 atmosphere. Supernatants were
harvested after 48 h, because kinetic studies previously indicated
that 48 h was the optimal time for assessing lymphokine production
after PHA stimulation (32, 33). Production of IL-2, IFN-, and IL-10
by PBMCs was evaluated with commercially available ELISA kits as
follows: IL-2, human IL-2 Intertest-2 (Genzyme, Cambridge, MA);
IFN-, human IFN- Intertest (Genzyme); and IL-10, human IL-10
Intertest 10 (Genzyme). To evaluate IL-12 production, PBMC (3 x
106/ml) were stimulated with Staphylococcus
aureus (SAC) for 48 h. Two commercially available kits
measuring either only the p70 heterodimer (Quantikine IL-12
Immunoassay, R&D Systems, Minneapolis, MN) or both the P70 heterodimer
and the P40 chains (total IL-12; Genzyme) were used to measure IL-12 in
the supernatants. All test kits were used following the procedures
suggested by the manufacturer. Cytokine production was calculated from
a standard curve of the corresponding recombinant human cytokine in
each case.
FACS analyzes
Leukocyte differentiation was performed using a Coulter counter (Coulter Electronics, Inc., Miami Lakes, FL). Lymphocyte and monocyte subsets were evaluated by flow cytometric analysis, using 50 µl of EDTA peripheral blood incubated for 30 min at 4°C with fluorochrome-labeled mAbs. After incubation, erythrocyte lysis and fixation of marked cells were performed using the Immuno-Prep EPICS Kit (Coulter Electronics) and Q-prep Work Station (Coulter Electronics).
Monoclonal Abs
The following mAbs were used: anti-CD4 (T4) energy-coupled dye (ECD phycoerythrin (PE)-Texas Red; IgG1 isotype), anti-CD8 (ECD; T8; IgG1), anti-CD4 FITC (IgG1), anti-CD19 (B4) PE (RD1; IgG1), anti-CD14 (Mo2) RD1 (IgM), anti-CD7 (3A1) FITC (IgG2b), and their isotype controls MsIgG1-FITC, MsIgG1-RD1, MsIgG2b-FITC, MsIgM RD1, MsIgG1 ECD (Cyto-stat Coulter Clone, Coulter Corp.). The specific mAb for CD95 (Fas Ag) PE (IgG1), CD80 (B7-1) FITC (IgM), CD86 (B7-2) FITC (IgG1), isotype controls MsIgG1 FITC, MsIgG1 PE, MsIgM FITC (PharMingen San Diego, CA), and SLAM (A12) PE (IgG1; DNAX Research Institute, Palo Alto, CA; 1 µg of SLAM was incubated with 50 µl of EDTA peripheral blood) were also used.
Immunophenotypic analyses
The cytometric analyses of lymphocyte and monocyte subsets were performed using an EPICS XL flow cytometer (Coulter Electronics) equipped with a single 15-mW argon ion laser operating at 488 nm interfaced with a 486 DX2 IBM computer (IBM United Kingdom, U.K.). Multiparametric data were acquired for 5000 events and analyzed using Coulter System II software (Coulter Electronic). Orange-red fluorescence from PE and RD1 (FL2) was collected through a 575-nm longpass filter, green fluorescence from FITC (FL1) was collected through a 525-nm bandpass filter, and finally, fluorescence from ECD (FL3) was measured through a 620-nm bandpass filter. Data were collected using linear amplifiers for forward and side scatter and logarithmic amplifiers for FL1, FL2, and FL3. Samples were first run using single fluorochrome-stained preparations for color compensation. The lymphocytes were selectively analyzed using forward and side scatter properties; the monocytes were identified using the specific mAb Mo1 (CD14). The absolute number of labeled cells was estimated by multiplying the absolute number of cells per microliter by the percentage of labeled cells.
Serum concentration of sApo-1/Fas
The serum concentration of sApo-1/Fas was evaluated using a commercially available ELISA assay (Bender MedSystems, Vienna, Austria) following the procedures suggested by the manufacturer. The serum concentration (units per milliliter) was calculated from standard curves according to the instruction of the manufacturer.
Statistical analyses
Data have been summarized according to standard statistical tests; nonparametric descriptive statistics (median, interquartile range) were preferred to avoid assuming a definite theoretical distribution for each dataset. Nonparametric tests were performed to evaluate differences between MS patients, divided according to their clinical status (SMS or AMS), and HC. In particular, for every variable, a Kruskal-Wallis (KW) one-way analysis of variance was performed; if this analysis showed significant differences between the different groups of individuals, nonparametric subtests (Mann-Whitney U tests) were used to examine differences between MS patients as a whole and HC, between MS patients classified according to clinical status, and between AMS or SMS and HC. Possible relationships between different immunologic parameters were evaluated using nonparametric Kendall’s rank correlation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC from 48 MS patients (26 SMS and 22 AMS) and from 16 HC were stimulated with either influenza virus (Flu) or a pool of allogeneic PBMC (Allo), and IL-2 production was measured in a bioassay. These two different stimuli were used because we previously demonstrated that they activated different Th APC pathways (34), thereby allowing a better evaluation of subtle defects in the immune response. Flu and all soluble Ags are recognized by CD4 T lymphocytes upon presentation by autologous APC after Ag processing (34). Th function in response to alloantigens can use the same pathway as soluble Ags; alternatively, CD4 and CD8 T lymphocytes can be stimulated either by allogeneic peptides processed and presented in association with self MHC molecules or by direct recognition of non-self MHC molecules on the surface of the allogeneic lymphocytes (34). Because soluble and alloantigens were used to analyze Th defects in HIV infection, we used these same Ags in MS patients to allow comparison of these results with those obtained in previous studies.
The results are shown in Figure 1
,
A (Flu-stimulated IL-2 production; by KW, p
= 0.03) and B (Allo-stimulated IL-2 production; by KW,
p = 0.01). IL-2 production in response to both Ags was
statistically reduced in MS patients compared with that in controls
(Flu, p = 0.02; Allo, p = 0.01). When
data from MS patients were divided according to the two clinical
subsets, the differences between AMS and SMS and those between AMS
and HC did not reach statistical significance. Nevertheless, SMS
patients produced significantly reduced amounts of IL-2 upon
Flu and Allo stimulation compared with HC (Flu, p
= 0.01; Allo, p < 0.01).
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PBMC of 48 MS patients (26 SMS and 22 AMS) and of 53 HC were
stimulated in vitro with PHA for 48 h, and IFN-, IL-2, and
IL-10 production was measured with ELISA assays. The median values and
the interquartile ranges for each cytokine are shown in Figure 2 for both groups of MS patients and for
the HC (by KW: IL-2, p < 0.01; IFN-,
p < 0.01; IL-10, p < 0.01). The
production of the type 1 cytokines IL-2 and IFN- was significantly
increased in MS individuals compared with that in HC (IL-2,
p < 0.01; IFN-, p < 0.01), whereas
IL-10 production was reduced in MS patients compared with that in HC
(p < 0.01). Data were subsequently analyzed by
dividing the MS patients according to the clinical classification, and
the following results (median production) were observed: IL-2: SMS, 986
pg/ml; AMS, 1417 pg/ml; HC, 658 pg/ml (SMS vs AMS, p <
0.01; SMS vs HC, p < 0.01; AMS vs HC,
p < 0.01; Fig. 2A); IFN-: SMS,
697 pg/ml; AMS, 1098 pg/ml; HC, 315 pg/ml (SMS vs AMS,
p < 0.01; SMS vs HC, p < 0.01; AMS vs
HC, p < 0.01; Fig. 2B); and IL-10:
SMS, 537 pg/ml; AMS, 290 pg/ml; HC, 266 pg/ml (SMS vs AMS,
p < 0.01; SMS vs HC, p < 0.01; AMS vs
HC, not significant; Fig. 2C). Thus, different
cytokine profiles were observed in MS patient compared with controls.
These changes (increased type 1 cytokine and decreased IL-10
production) were exacerbated in patients suffering from an acute MS
episode.
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observed in AMS
and because IL-12 is a pivotal mediator of the effector phase of CMI,
we measured SAC-stimulated IL-12 production in the same patients and
controls. Because the metabolically active form of IL-12 is the p70
heterodimer, but both the p70 and the metabolically inactive p40 chains
can be secreted by cells (35), we used two different kits: one
measuring only p70 and the second measuring both the p70 heterodimer
plus the p40 chains (total IL-12). The results are shown in Figure 3 and indicate that SAC-stimulated p70
production was comparable in MS and HC and in AMS and SMS patients
(Fig. 3A; not significant by KW). In contrast to these
results a more complex pattern was obtained when SAC-stimulated
production of total IL-12 was examined (by KW, p <
0.01). To rule out the possibility that these results stemmed from
differences in the relative percentage of CD14+ cells in
the samples, we analyzed the percentage of CD14+ monocytes
in all AMS and SMS patients and in HC. Results showed the percentages
of these cells to be comparable in the three groups (data not shown).
Thus, total IL-12 production was 1) significantly augmented in SMS
compared with that in AMS patients (p < 0.01),
and 2) significantly reduced in AMS compared with HC
(p = 0.02; Fig. 3B).
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Lymphocytes.
The total number of lymphocytes, the percentage of
CD4+ and CD8+ T lymphocytes, and the
percentage of CD4+ T lymphocyte coexpressing SLAM or CD7
(two cell surface markers suggested to be expressed by T lymphocytes
that preferentially produce type 1 cytokines upon restimulation) were
analyzed in PBMC of MS patients and HC. Results are shown in Table I and indicate that whereas absolute
lymphocyte count and the percentage of CD8+ T lymphocyte
did not differ between MS patients and controls, CD4+ T
lymphocytes were significantly increased in MS patients compared with
controls (p = 0.03). More interestingly, the
percentages of both CD4+SLAM+ T lymphocyte and
CD4+CD7+ T lymphocyte were increased in MS
compared with those in HC (CD4+SLAM+,
p = 0.01; CD4+CD7+,
p < 0.01). No differences were observed when
CD4+SLAM+ and CD4+CD7+
T lymphocytes were compared in AMS and SMS patients (Table II). The
percentages of CD4+SLAM- and
CD4+CD7- lymphocytes were also examined. The
results suggested a trend such that the percentage of both lymphocyte
subsets was decreased in MS patients compared with controls, although
this trend did not reach statistical significance (data not shown). We
also verified that the presence of CD4+SLAM+
and CD4+CD7+ T lymphocytes was statistically
correlated. Thus, a positive correlation between expression of the two
Th1-defining subsets (Spearman correlation coefficient (SCC) = 0.0394;
p < 0.01) was detected. Additionally,
CD4+SLAM+ lymphocytes were negatively
correlated with both CD4+SLAM- (SCC =
-0.0349; p < 0.01) and
CD4+CD7- (SCC = -0.0332;
p < 0.02) lymphocytes (data not shown).
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The serum concentration of sApo-1/Fas was measured in MS patients
and controls. The median values and the interquartile ranges for each
cytokine are shown in Figure 4 for both
groups of MS patients and for the HC (by KW, p = 0.01).
Whereas the median serum concentration of sApo-1/Fas was comparable
between SMS and HC, a significantly augmented concentration of this
molecule was detected in AMS compared with that in both SMS
(p = 0.01) and HC (p <
0.01).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We observed that 1) Ag-stimulated TH function was defective as assessed
by IL-2 production, and 2) mitogen-stimulated type 1 cytokine
production was increased and type 2 cytokine production was reduced in
MS patients compared with those in HC. Mitogen-stimulated IFN- and
IL-2 production was increased and IL-10 production was decreased in AMS
patients compared with those in SMS patients, suggesting that different
cytokine profiles are associated with acute (higher type 1 cytokine) or
stable (higher IL-10) disease. These results are remarkably similar to
those obtained by Correale et al. (36), who analyzed cytokine
production by proteolipid protein-specific T cell clones obtained from
patients either during acute episodes of disease or during the
remission phase, and from controls.
Although SAC-stimulated production of the metabolically active IL-12
p70 heterodimer was similar in AMS, SMS, and controls, the production
of total IL-12 was significantly increased in SMS compared with AMS
patients and significantly reduced in AMS patients compared with HC,
suggesting an alteration in the production of the metabolically
inactive p40 chains in MS patients. The p40 homodimer subunit of IL-12
has been shown to be an antagonist of the biologic effects of IL-12 in
vitro (37) and was recently demonstrated to elicit a potent
immunosuppressive effect on Th1-mediated immune functions (rejection of
allografts) in vivo (38) and to directly antagonize IL-12-dependent
IFN- responses in murine endotoxic shock (37). We have not directly
quantified the presence of p40 homodimers in the supernatants; it is
nevertheless tempting to speculate that the augmented production of
total IL-12 observed in SMS patients could include increased quantities
of p40 homodimers, allowing for a regulatory mechanism controlling type
1 cytokine production and activation of CMI. Increased IL-12 p40 chain
was reported by others in acute MS plaques; these findings were
interpreted as supporting a direct involvement of IL-12 in MS (39). We
consider the pathogenic role of IL-12 in MS to be secondary to the
ratio of IL-12 p70 to p40. Thus, overproduction of the nonfunctional
IL-12 p40 homodimer could be present and involved in dampening
cell-mediated immunity in SMS patients, whereas reduced production of
this chain could be involved in increasing potentially pathologic
responses in acute episodes of MS. In this regard it is important to
underline that a therapeutic use of the p40 homodimer of IL-12 in acute
MS was recently hypothesized (40).
The percentage of CD4-expressing T lymphocytes was significantly
increased in MS patients compared with controls, and as previously
reported, CD8 cells tended to be reduced in the same patients (41, 42, 43, 44).
Because type 1 cytokine secretion was also augmented in MS patients and
because markers suggested to be associated with the preferential
production of type 1 cytokines upon restimulation (25, 45) are now
available, we tested whether the increased percentage of CD4 could be
secondary to the expansion of lymphocytes expressing such markers.
Phenotypical analysis confirmed the cytokine data, as the percentage of
SLAM-expressing lymphocyte was increased in MS patients compared with
that in HC. Engagement of SLAM up-regulates IFN- production by CD4 T
lymphocyte upon restimulation (25, 45). Because IFN- production in
MS patients was augmented compared with that in controls, we
hypothesize that this increase was due to
CD4+SLAM+ T lymphocytes. That an
imbalance in the percentage of lymphocyte-expressing markers, which was
suggested to be associated with Th1 and Th2, is present in MS was
further underlined by the observations that 1) optimally proliferating
and IL-2-secreting CD4+CD7+ lymphocytes (26)
were augmented in MS compared with control values; and 2)
SLAM- and CD4+CD7- T lymphocytes
were reduced in MS patients compared with those in controls. The
strength of these data was underlined by the observation that the
detection of SLAM+ T lymphocytes was correlated positively
to that of CD4+CD7+ lymphocyte and negatively
to the detection of SLAM- and
CD4+CD7- cells. Thus, clusters of
differentiation suggested to correlate with different functional
abilities of immune cells are expressed in a discreet and
characteristic way in MS patients. On the other hand, although
increased production of IL-2 and IFN- was observed in AMS compared
with SMS patients, this augmented cytokine production was not reflected
in statistical differences in the percentage of T lymphocytes
expressing either SLAM or CD7+. This discrepancy could be
due to a number of reasons, currently under investigation, including 1)
the presence in AMS patients of one or more type 1 cytokine-secreting
populations not expressing either SLAM or CD7, 2) an increased
secretion of type 1 cytokines on a per cell basis in AMS patients, and
3) the expansion in these patients of a type 1 cytokine-secreting
T lymphocyte population.
Because the interaction between CD28 and B7-1 was suggested to preferentially stimulate type 1 cytokine production, whereas CD28 triggering of B7-2 should mainly activate the generation of type 2 cytokines (46, 47), we examined B7-1 and B7-2 expression in monocytes of our patients. It is nevertheless important to underline that the expression of B7-2 is induced earlier than that of B7-1 in most acute and chronic immune responses, with the noticeable exception of murine allergic encephalomyelitis (48, 49), a classic animal model for human MS (50). A significant increase in the percentage of B7-2-expressing monocyte was detected in MS patients compared with HC, whereas no differences were observed in the expression of B7-1 despite the fact that both type 1 cytokine production and the percentage of Th1 markers expressing lymphocytes were augmented in such patients. The increased expression of B7-2 observed in our patients is apparently in contrast with these findings and could be explained as a regulatory loop with which the hyperactivated immune response is attempting to down-modulate itself. Other experiments will nevertheless be necessary to clarify this result.
The interaction between membrane-expressed Apo-1/Fas and its ligand is known to trigger apoptosis (30, 51, 52, 53), and the secretion of a soluble form of Fas receptor (sApo-1/Fas) was recently suggested to be a mechanism by which cells escape apoptosis (31, 54). Alterations of this system resulting in accelerated/augmented apoptosis are known to occur in HIV infection (55, 56) and in drug-induced apoptosis of leukemia cells (57). In particular, in HIV infection both overexpression of Apo-1/Fas (58, 59, 60) and a reduced serum concentration of sApo-1/Fas were observed4 and were proposed as prognostic markers of disease progression. Apoptosis of Ag T cells was hypothesized as a mechanism leading to death of autoreactive T lymphocytes, thus limiting the duration of acute episodes of disease. We analyzed the expression of Apo-1/Fas on immune cells and the serum concentration of sApo-1/Fas in MS patients and observed that the percentage of Apo-1/Fas-expressing immune cells is increased in MS patients compared with that in controls and in AMS compared with that in SMS patients and that augmented expression of Apo-1/Fas is mostly evident on the surface of macrophages. Because macrophage-induced, Fas-dependent apoptosis in HIV infection is an important mechanism leading to apoptosis of CD4+ and CD8+T lymphocytes (61, 62), the up-regulation of the Apo-1/Fas receptor present in MS patients (and most evident in AMS) could lead to the activation of self-regulatory, apoptosis-based loops in the attempt to eliminate autoreactive lymphocytes. An increment in the serum concentration of sApo-1/Fas, a mediator suggested to be capable of preventing apoptosis, was observed in AMS patients. This confirms the dysregulation of the nerve growth factor/TNF receptor family we observed in MS and could, at least theoretically, be involved in disease relapse by preventing apoptosis of autoreactive T cell clones. More detailed analyses of susceptibility to apoptotic death in different phases of the disease and of possible correlations with the various components of the TNF receptor family will be necessary to clarify these findings.
In conclusion, a complex alteration of the immune response is present in MS. This alteration includes two phenomena that to our knowledge are reported here for the first time in human pathology: 1) the augmented percentage of SLAM-expressing lymphocytes, and 2) the dissociation between the production of the p70 and p40 components of IL-12. Immune defects in MS patients can be detected both functionally (impaired cytokine production) and phenotypically, and could be summarized as an attempt on the part of an hyperactivated immune system to reduce its abnormal activation either by producing factors known to suppress immune responses (IL-10 and p40 heterodimers) or by modulating the susceptibility of immune cells to apoptosis. Other detailed and possibly longitudinal analyses will be needed to ascertain the importance of these observations within a clinical and therapeutical setting.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Prof. Mario Clerici, Cattedra di Immunologia, Università degli Studi di Milano, Via Venezian 1, 20133 Milan, Italy. E-mail address:
3 Abbreviations used in this paper: MS, multiple sclerosis; CMI, cell-mediated immunity; AMS, acute multiple sclerosis; SMS, stable multiple sclerosis; sApo-1, soluble Apo-1; RR, relapsing-remitting; HC, healthy controls; SAC, Staphylococcus aureus; ECD, energy-coupled dye; PE, phycoerythrin; KW, Kruskal-Wallis; Flu, influenza virus; Allo, allogeneic peripheral blood mononuclear cells; SCC, Spearman correlation coefficient; SLAM, signaling lymphocytic activation marker.
4 F. J. Medrano, M. Leal, D. Arienti, C. Rey, A. Zagliani, Y. Torres, E. Lissen, and M. Clerici. 1997. TNF ß and soluble Apo-1/Fas independently predict progression to AIDS in HIV seropositive patients. Submitted for publication.
Received for publication June 25, 1997. Accepted for publication October 22, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J. R. Bleharski, K. R. Niazi, P. A. Sieling, G. Cheng, and R. L. Modlin Signaling Lymphocytic Activation Molecule Is Expressed on CD40 Ligand-Activated Dendritic Cells and Directly Augments Production of Inflammatory Cytokines J. Immunol., September 15, 2001; 167(6): 3174 - 3181. [Abstract] [Full Text] [PDF]
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X. Wang, M. Chen, K. P. Wandinger, G. Williams, and S. Dhib-Jalbut IFN-{beta}-1b Inhibits IL-12 Production in Peripheral Blood Mononuclear Cells in an IL-10-Dependent Mechanism: Relevance to IFN-{beta}-1b Therapeutic Effects in Multiple Sclerosis J. Immunol., July 1, 2000; 165(1): 548 - 557. [Abstract] [Full Text] [PDF]
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C Rohowsky-Kochan, D Molinaro, A Choudhry, M Kahn, and S D Cook Impaired interleukin-12 production in multiple sclerosis patients Multiple Sclerosis, October 1, 1999; 5(5): 327 - 334. [Abstract] [PDF]
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), 22
patients with AMS (•), and 16 HC (
). A shows results
from Flu-stimulated PBMC; in B, IL-2 production by PBMC
stimulated with a pool of irradiated Allo is shown. In A and
B, each dot represents IL-2 production by one individual,
whereas the bars indicate median values. Statistically significant
differences are shown: * indicates p = 0.02; **
indicates p = 0.01.
