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The Journal of Immunology, 2002, 168: 4293-4300.
Copyright © 2002 by The American Association of Immunologists

Attenuation of Experimental Allergic Encephalomyelitis in Complement Component 6-Deficient Rats Is Associated with Reduced Complement C9 Deposition, P-Selectin Expression, and Cellular Infiltrate in Spinal Cords1

Giang T. Tran2,*, Suzanne J. Hodgkinson2,3,*, Nicole Carter*, Murray Killingsworth{dagger}, S. Timothy Spicer* and Bruce M. Hall*

Departments of * Medicine, and {dagger} Anatomical Pathology, University of New South Wales, Liverpool Hospital, Liverpool, New South Wales, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of Ab deposition and complement activation, especially the membrane attack complex (MAC), in the mediation of injury in experimental allergic encephalomyelitis (EAE) is not resolved. The course of active EAE in normal PVG rats was compared with that in PVG rats deficient in the C6 component of complement (PVG/C6-) that are unable to form MAC. Following immunization with myelin basic protein, PVG/C6- rats developed significantly milder EAE than PVG/C rats. The anti-myelin basic protein response was similar in both strains, as was deposition of C3 in spinal cord. C9 was detected in PVG/C rats but not in PVG/C6-, consistent with their lack of C6 and inability to form MAC. In PVG/C6- rats, the T cell and macrophage infiltrate in the spinal cord was also significantly less than in normal PVG/C rats. There was also reduced expression of P-selectin on endothelial cells, which may have contributed to the reduced cellular infiltrate by limiting migration from the circulation. Assay of cytokine mRNA by RT-PCR in the spinal cords showed no differences in the profile of Th1 or Th2 cytokines between PVG/C and PVG/C6- rats. PVG/C rats also had a greater increase in peripheral blood white blood cell, neutrophil, and basophil counts than was observed in the PVG/C6-. These findings suggest that the MAC may have a role in the pathogenesis of EAE, not only by Ig-activated MAC injury but also via induction of P-selectin on vascular endothelium to promote infiltration of T cells and macrophages into the spinal cord.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Experimental allergic encephalomyelitis (EAE) is an animal model of multiple sclerosis. Both EAE and multiple sclerosis are considered to be T cell-mediated diseases (1), as these cells can break down the blood brain barrier (BBB) and cause inflammation and demyelination in the CNS (2). The major mechanism of injury to the CNS is thought to involve infiltration of CD4+ T cells, which leads to release of inflammatory cytokines and promotes macrophage infiltration and activation. CD4+ cells of the Th1 subset that produce IL-2, IFN-{gamma}, and TNF-{beta} are thought to directly mediate CNS injury (3) but also promote Ig switch to complement-fixing isotypes. Th2 cells are believed to down-regulate disease (4) and can switch Ig to non-complement-fixing isotypes. The role of complement activation in the pathogenesis of EAE is less clear, but several studies suggest that complement activation may also contribute to the development of clinical EAE (5, 6).

After IgM or complement-fixing IgG isotypes bind to Ag, they can activate complement via the classical pathway (7, 8). Complement activation also occurs via lectins or the alternate pathway (7, 9, 10). All three pathways lead to activation of C3, which aids Ag presentation to B cells, thereby facilitating T cell-dependent Ig responses. Activated C3b lyses C5 to promote C5a anaphylatoxin and C5b, which triggers the formation of the membrane attack complex (MAC) by cascade of activation of C6, C7, and C8, which leads to C9 activation and polymerization. The functions of MAC include direct lysis of pathogens or cells, promotion of phagocytosis, and production of anaphylatoxin, as well as activation of vascular endothelium to promote cellular infiltration (7, 8).

Cobra venom factor (CVF), which blocks the complement cascade at the level of C3, delays the onset of EAE as well as reducing demyelination, without decreasing cellular infiltration (5, 11, 12, 13). Likewise, in experimental allergic neuritis, an animal model of acute inflammatory demyelinating peripheral neuropathy (14), CVF treatment reduces disease severity and demyelination (15, 16). Reduced severity and demyelination have been observed in C3 knockout mice when EAE is induced by immunization with myelin oligodendrocyte glycoprotein (MOG)p35–55 (17). Using a higher dosage of MOGp35–55 in a different strain of C3 knockout mice, there was no reduction of severity of EAE (18). In the C3 knockout with milder EAE, there was a reduced macrophage, T cell infiltrate, and ICAM-1 expression in the CNS. In this model, demyelination is Ab dependent, and the reduced infiltrate was attributed to reduced production of C3a and C5a. Both C3a and C5a can directly chemoattract monocytes and T cells, induce chemokine production by other cells, and increase expression of adhesion molecules on vascular endothelium. Factor B knockout mice also develop less severe EAE with reduced infiltration of T cells, macrophages, and ICAM-1+ cells (17). C3 activation by Ag has multiple effects, including facilitating Ag presentation that is required for activation of T cell responses (19, 20). Whether the reduced severity of EAE in C3 knockouts was due to reduction of Ag presentation and the consequent immune response or to a failure to form MAC is not resolved. These studies did not directly examine the function of components that are activated after C5a, in particular the formation of lytic MAC.

In EAE, MAC deposition has been demonstrated on the myelin sheath (21, 22). Furthermore, only complement-fixing isotypes of anti-MOG mAbs induce EAE and demyelination (23, 24). In vitro treatment with complement regulatory molecule CD59, which inhibits complement, protects rat oligodendrocytes from complement-induced lysis while neutralization of CD59 renders them susceptible to lysis (25). Furthermore, transgenic mice expressing complement inhibitor Crry in the CNS do not develop MOG peptide-induced EAE (26). These studies are consistent with the possibility that MAC-mediated injury may be an important pathogenic mechanism in EAE.

This study examined the role of the MAC in the pathogenesis of active EAE by comparing disease in normal PVG rats and in rats that are deficient in C6 (PVG/C6-) that are unable to form MAC (27). These rats have normal C3 and C5, which allow us to examine the role of C6, C7, C8, and C9 in EAE. PVG/C6- rats developed milder disease with reduced macrophage and T cell infiltrate, as well as reduced induction of the vascular adhesion molecules ICAM-1 and P-selectin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

The complement-deficient PVG (PVG/C6-) rats were originally derived from Bantin & Kingman (Fremont, CA) (27) and bred in the animal house at Liverpool Hospital (Liverpool, New South Wales, Australia) as described (28). PVG and Sprague Dawley rats were also bred at Liverpool Hospital. To determine that our strain of PVG/C6- rats have the same genetic background as our PVG/C strain, skin grafts were exchanged between PVG/C and PVG/C6- rats and were accepted indefinitely (>200 days). This indicated genetic identity apart from C6 deficiency. In all experiments, 10- to 12-wk-old female PVG/C and PVG/C6- rats were used.

Hemolytic complement assay

Rat anti-human RBC was produced by IP inoculation of 1–2 x 108 RBC into female Sprague-Dawley rats for three doses at three weekly intervals before bleeding for sera at 6 wk. Briefly, rat serum samples were diluted in TBS (0.05% gelatin, 0.15 M CaCl2, 0.5 M MgCl2, 0.12 M NaCl, 0.28% triethanolamine, and 0.017 M HCl) and incubated with 6% human RBC for 30 min at 37°C. Hemolysis was measured by reading the absorbance (540 nm) of the supernatant obtained after centrifugation (Bio-Rad, Hercules, CA). To confirm that PVG/C6- were deficient only in the C6 component, their serum was reconstituted with 60 µg/ml purified human C6 (Sigma-Aldrich, St. Louis, MO), which restored its hemolytic activity. This amount is equivalent to the C6 concentration detected in the PVG/C rat serum (29).

Induction of disease and clinical assessment

PVG/C and PVG/C6- rats were immunized with 50 µg guinea pig myelin basic protein (MBP) emulsified with IFA, and 5 mg/ml heat-killed Mycobacterium tuberculosis (Difco, Detroit, MI) was injected s.c. in the hind footpads. Clinical disease was assessed and graded as follows: 0, no abnormality; 1/2, partial loss of tail tone; 1, complete loss of tail tone; 2, hind limb weakness; 3, hind limb paralysis and front limb weakness; 4, paraplegic; 5, moribund. The final score for each group is reported as a mean ± SD.

Blood counts

Whole blood was collected from the tail vein at days 0, 7, 14, 21, 28, and 35 in a Minicollect tube containing K3EDTA (Greiner Labortecnik, Kremsmünster, Austria). Samples were analyzed on a Celldyn 3500 (Diagnostic, San Francisco, CA) programmed for rat peripheral RBCs. The absolute number of white blood cells (WBC), lymphocytes, monocytes, neutrophils, basophils, and eosinophils was expressed per milliliter of blood.

Histopathology and immunostaining

At least three rats in each group were sacrificed to collect spinal cord and popliteal lymph nodes at days 14 and 18 postimmunization. Rats were perfused with PBS by cardiac puncture before spinal cord sections were removed and embedded in Tissue-Tek O.C.T. compound (Miles, Elkhart, IN). The cervical spinal cord immediately below the brain stem was taken and this sample was divided; half was used for histology and the other half for mRNA extraction for RT-PCR studies. Sections of cervical spinal cord (5-µm thickness) were cut on a cryostat, air-dried, and fixed in acetone for 10 min before performing a three-step indirect immunoperoxidase technique as described (30). The mAbs used for the first step were R7.3 (anti-TCR{alpha}{beta} chain), W3/25 (anti-CD4 on T cell subset and macrophages), OX-12 (anti-rat Ig k-chain), OX-62 (anti-dendritic cells), IA29 (anti ICAM-I, CD54) (all from BD PharMingen, San Diego, CA), anti P-selectin polyclonal (anti-CD62P; a gift from Dr. M. Berndt, Baker Research Institute, Parkville, Victoria, Australia), ED1 (anti-macrophage; Serotec, Oxford, U.K.), C9 (rabbit anti-rat C9 polyclonal; a gift from Dr. S. Piddlesden, Sanofi-Synthelabo, Sydney, Australia) (31), and GARa (sheep anti-rat C3; Nordic Immunology, Tilburg, The Netherlands). Stained cells were counted as cells per high-powered field (magnification, x40). Data from multiple spinal cord samples were expressed as mean ± SD. Staining of C3, C9, P-selectin, and ICAM-1 were assessed using a semiquantitative scale as follows: 0, no staining; +, weak patchy staining; ++, strong patchy staining; +++, widespread weak staining; ++++, widespread strong staining.

Semiquantitative PCR

Popliteal lymph nodes and spinal cord sections were removed at days 14 and 18 postimmunization and snap-frozen in liquid nitrogen. mRNA extraction and cDNA synthesis were performed on these samples as previously described (32, 33). Specific primers for rat IL-2, IL-10, IL-4, IL-12R{beta}2, IFN-{gamma}, TNF-{alpha}, and TGF-{beta} were described elsewhere (32, 34). ICAM-1 sense (5'-ttgagaactgtggcaccacgagt-3') and antisense (5'-ctgacctcggagacattcttgaa-3'), P-selectin sense (5'-cgaaagatcaacaataagtggac-3') and antisense (5'-ggtagcaggagcaggtgtagct-3'), Fas sense (5'-ctgcacaacaagtggaggtgca-3') and antisense (5'-tcggcagttctccagatgta-3'), and Fas ligand (FasL) sense (5'-actgggtagacagcagtgccac-3') and antisense (5'-gttaagagggccacactccttgg-3') were designed from rat cDNA sequence or from areas of homology between the mouse and human genes.

Methods for RNA extraction, reverse transcription, and PCR for cytokines have been described (34, 35). cDNA samples were serially diluted in diethyl pyrocarbonate-treated water as neat, 1/10, 1/20, 1/40, and 1/80. PCR used 1 µl of cDNA, 0.5 U of Taq polymerase (Biotech International, Perth, Australia), 1.5 mM MgCl2, and 125 µM dNTP (Promega, Madison, WI).

All samples were assayed using GAPDH primers, a housekeeping gene, to confirm that the concentration of template cDNA was uniform in all samples. Standard PCR conditions consisted of an initial 3-min denaturation (at 94°C) followed by 30 s each of denaturation (at 94°C) and primer annealing (at 60°C) cycles, a 50-s extension (at 72°C) cycle, then a final extension of 72°C for 4 min, performed on a Corbett Thermal Cycler (Corbett Research, Sydney, Australia). The number of cycles for each primer set was between 23 and 35 cycles and was determined to ensure the dilutions were tested on the linear phase of amplification. A quarter of total PCR products were run on 6% polyacrylamide gels with pUC19/HpaII digested m.w. marker, stained with ethidium bromide, and photographed under UV light using a Kodak DC40 camera and Digital Science software (Kodak, Rochester, NY).

ELISA

Serum from PVG/C and PVG/C6- rats collected at days 0, 7, 14, and 21 was assayed for total Ig, IgG1, IgG2a, and IgG2b isotype responses against MBP. Briefly, MBP at a concentration of 25 µg/ml in a buffer of 0.15% Na2CO3 and 0.29% NaHCO3 (pH 9.5) was incubated on a 96-well plate overnight at 4°C. The plates were washed twice with water and soaked for 5 min with buffer before triplicate samples of serum at a 1/200 dilution were added and incubated for 2 h. The plates were then washed twice with buffer before being reacted with HRP-rabbit anti-rat Ig (DAKO, Copenhagen, Denmark) diluted at 1/2000 with 10% human sera in PBS (pH 7.2) for 1 h at 37°C. The plates were then washed two times with buffer before reaction with phosphatase substrate (Sigma-Aldrich). The reactions were read at 20 min on a microplate reader (Bio-Rad) at 450 nm wavelength.Positive control anti-MBP serum with high titer were collected at day 60 from Lewis rats that had recovered from active EAE and had been reimmunized with IFA-MBP at days 30 and 44. Positive and negative control serum samples from normal Lewis rats were included on all plates. Data are expressed as absorbance at 450 nm.

Statistics

Data on clinical score and weight loss were analyzed in two ways. First, the maximal disease score and maximal weight loss for each rat at any time, as well as day of onset and recovery, were compared by the Student two-tailed t test. Second, the clinical scores on each day postimmunization were compared using nonparametric Wilcoxon signed rank test. Student’s t tests were used for all cell counts and Ab titers. Values of p <= 0.05 were considered a significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Active EAE in PVG/C and PVG/C6- rats

In five separate experiments, 95% of PVG/C rats developed clinical signs, with a mean maximal clinical score of 2.3 ± 0.9 (n = 46). Only 70% of PVG/C6- rats immunized with the same batches of MBP/CFA developed symptoms. Their mean maximal clinical score was significantly less: 1 ± 0.8 (n = 44; p < 0.0001). Clinical severity in PVG/C6- rats was significantly lower than PVG/C rats at all data points observed during the clinical phase. Also, the time of onset of clinical disease was significantly delayed in the PVG/C6- rats (14.5 ± 2.2 days (n = 32) compared with 13 ± 1.1 days in PVG/C rats (n = 43; p = 0.001)). Clinical signs persisted significantly longer in the PVG/C rats (5 ± 1.8 days; n = 37) as compared with PVG/C6- (3 ± 2.4 days; n = 29; p = 0.0001). Mean maximal percentage weight loss during the clinical phase was significantly greater in PVG/C (9.1 ± 6.3%; n = 37) compared with PVG/C6- (5.4 ± 5.8%; n = 44; p = 0.009). There were also significant differences in weight loss at all data points taken during the clinical phase. All five experiments conducted showed similar results. These data are expressed in a different form in Fig. 1Go, which shows the mean clinical score (Fig. 1GoA) and mean weight loss (Fig. 1GoB) on each day. As the peak of clinical activity in individual rats occurs between days 14 and 18, the mean clinical score on an individual day was less than the mean maximal clinical score. Both clinical score and weight loss were significantly less in PVG/C6- from day 12 to 19 and from day 13 to 18, respectively. Exclusion of the 5% of PVG/C and 30% of PVG/C6- rats that did not develop clinical disease from the analysis still demonstrated a milder maximal disease in the PVG/C6- (1.5 ± 0.5; n = 31) than in PVG/C (2.4 ± 0.8; n = 44; p < 0.001). Delayed onset and reduced weight loss were also significant when data from unaffected rats were excluded. Thus, the differences were not only due to the lower rate of induction of EAE in PVG/C6-.



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  		FIGURE 1. Comparison of mean clinical disease recorded daily during the course of EAE in PVG/C ({circ}, n = 46) and PVG/C6- rats ({blacksquare}, n = 44). Shown are combined data from five separate experiments. A, Clinical score, which was significantly less in PVG/C6- than in PVG/C at days 12 (p < 0.005), 13 (p < 0.0001), 14 (p < 0.0001), 15 (p < 0.0001), 16 (p = 0.0001), 17 (p = 0.005), 18 (p = 0.004), and 19 (p = 0.04). B, Weight loss, which was significantly less in the PVG/C6- rats compared with PVG/C rats at days 13 (p < 0.01), 14 (p < 0.001), 15 (p = 0.001), 16 (p = 0.005), 17 (p < 0.01), and 18 (p = 0.05).

 
Anti-MBP responses in PVG/C and PVG/C6- rats

Serum was collected at weekly intervals from the day of immunization to 5 wk postimmunization and was examined for Abs (total Ig, IgG1, IgG2a, and IgG2b) against MBP. Abs against MBP were detected in both groups at day 14 postimmunization, including elevation in the levels of IgG1, IgG2a, and IgG2b (Fig. 2Go). Anti-MBP levels remained relatively high up to day 35, after the animals had completely recovered. There were no differences in total amounts of Ig, IgG2a, or IgG2b between PVG/C (n = 8) and PVG/C6- (n = 7). However, compared with PVG/C there were reduced levels of IgG1 in PVG/C6- animals (Fig. 2Go) at days 14 and 21 and a borderline reduction at day 28 (p = 0.01, 0.02, and 0.07, respectively).



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  		FIGURE 2. Total Ig, IgG2a, and IgG2b responses against MBP were determined by ELISA. Comparison between PVG/C ({blacktriangleup}, n = 8) and PVG/C6- rats ({blacksquare}, n = 7) indicated no significant differences in total Ig, IgG2a, and IgG2b between the two groups. Less IgG1 was detected in PVG/C6- rats at days 14 and 21, and a borderline reduction was detected at day 28 (p = 0.01, 0.02, and 0.07, respectively). The result is expressed as mean of OD450 ± SD.

 
Peripheral blood cell counts

Peripheral blood counts on PVG/C6- (n = 12) and PVG/C (n = 12) rat were compared at days 0 (before immunization), 7, 14, 21, 28, and 35. Prior to immunization, all parameters of the white cell count in PVG/C6- rat were not different from those in PVG/C. At day 7 there was a significant increase in absolute cell count in WBC, neutrophils, monocytes, and basophils, but no significant change in eosinophils (Fig. 3Go). These changes persisted throughout the study period in PVG/C. In PVG/C6-, the cell count returned rapidly to normal and was significantly less than PVG/C at most time points after day 14. The lymphocyte count was reduced in all groups during the preclinical and effector phase (days 7 and 14) and returned to normal levels for the remainder of the study period (Fig. 3Go). This is consistent with previous reports of a reduction of peripheral CD4+ and CD8+ T cells observed during the preclinical phase (36, 37, 38). Our data are different from previous studies, which have shown no change in the frequency or absolute numbers of monocytes and basophils in the recovery phase in EAE with normal complement (37). In both normal PVG/C and PVG/C6-, there was initial monocytosis at day 7 which returned to normal at day 14. However, only the PVG/C had a second phase of monocytosis, which may have been associated with the recovery phase of the CNS inflammation or due to sublytic MAC activity.



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  		FIGURE 3. Comparison of peripheral blood cell count responses between PVG/C ({blacksquare}) and PVG/C6- ({square}) after immunization with MBP. An increased number of circulating WBC, neutrophils, monocytes, and basophils was detected in all groups (n = 8) after immunization. Significantly higher levels of WBC, neutrophils, and basophils were detected in PVG/C compared with PVG/C6- rats at many time points, even after recovery from clinical disease. Lymphocyte counts were significantly reduced in both PVG/C and PVG/C6- at 7 and 14 days, then returned to normal. *, p <= 0.05; **, p <= 0.01; ***, p <= 0.001; ****, p <= 0.0001

 
Examination of the response in the regional node to the site of immunization

There was no difference in LN weight between PVG/C (64.1 ± 15 mg; n = 6) and PVG/C6- (60.8 ± 6.5 mg; n = 6; p = NS) rats. Popliteal LN taken at day 14 showed comparable expression of IL-2, IL-4, IL-10, TGF-{beta}, IFN-{gamma}, Fas, FasL, and IL-12R{beta}2 mRNA in PVG/C and PVG/C6- rats (Fig. 4GoA). Three nodes from each group were compared with consistent results; thus, only one sample is shown in Fig. 4Go. Samples taken at day 18 also show a similar level of mRNA expression for all cytokines examined (Fig. 4GoB). Taken together, these results suggested that there is similar recruitment and activation of lymphocytes in the node draining the site of immunization and that the primary response to MBP in the two groups is similar. Failure of immunization does not explain the improved clinical status of the PVG/C6- rats.



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  		FIGURE 4. Semiquantitative RT-PCR of cytokines in the popliteal LNC. As the results are similar, only one representative of the three samples is shown. Detection of GAPDH mRNA at low PCR cycles, with identical band intensity at the highest dilution factor, confirms that a similar amount of mRNA/cDNA was used in all PCR. RT-PCR of cytokine mRNA was conducted at days 14 (A) and 18 (B) postimmunization. PCR results are expressed as the dilution factor of the cDNA template at which a specific PCR condition can be detected (see Materials and Methods). The results show no preferential pattern of Th1 or Th2 expression between the PVG/C and PVG/C6- groups (n = 3).

 
Examination of the cellular inflammatory response in spinal cords

Comparison of the mononuclear infiltrate was made in spinal cord samples from PVG/C and PVG/C6- at days 14 and 18 postimmunization. At days 14 and 18 there were significantly more mononuclear cells including macrophages, TCR{alpha}{beta}, CD4+ cells, B cells, and dendritic cells in the spinal cords of PVG/C compared with the PVG/C6- samples (Table IGo). Most of the infiltration was detected in the perivascular area (illustrated in Fig. 5Go). Normal PVG/C and PVG/C6- spinal cords had no mononuclear cell (data not shown).


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  		Table I. Comparison of immunostaining of cells, complement components, and adhesion molecules in the spinal cords at days 14 and 18 postimmunization in PVG/C and PVG/C6- rats1

 


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  		FIGURE 5. Immunoperoxidase staining of P-selectin, ICAM-1, and C9 in the CNS of PVG/C and PVG/C6- rats actively immunized with MBP. The samples were taken at day 14. A, PVG/C6- showed no detectable level of C9 component and very little expression of P-selectin and ICAM-1 around vascular tissues, all of which were readily observed in PVG/C rats. Similar C3 deposits were observed in PVG/C6- and PVG/C rats. B, Staining of macrophages (ED1), TCR{alpha}{beta}+ cells (R73), and CD4+ cells (W3/25) showed a markedly reduced infiltrate of all cell types at day 14 in PVG/C6- compared with PVG/C rats.

 
The level of mRNA for Th1 (IL-2, IL-12R{beta}2, IFN-{gamma}) and Th2 (IL-4, IL-10, TGF-{beta}) cytokines as well as Fas and FasL was assessed at days 14 and 18 postimmunization in the spinal cord. Three samples from each group were analyzed at both time points. As all three within each group had identical RT-PCR products, only one is shown in Fig. 6Go. In the control spinal cords of CFA-immunized rats and normal rats, mRNA for these cytokines was not detected by RT-PCR, except for low levels of TGF-{beta} (data not shown). Thus, detection of mRNA was consistent with increased cytokine mRNA levels, most likely in the infiltrating mononuclear cells. At day 14, comparable up-regulation of TNF-{alpha}, IFN-{gamma}, TGF-{beta}, Fas, and IL-4 was observed in both groups (Fig. 6GoA). However, there were 2-fold greater levels of IL-2, IL-10, and IL-12R{beta}2 in the PVG/C compared with PVG/C6- rats. By day 18, the levels of cytokines had fallen in the PVG/C rats, consistent with the reduced mononuclear infiltrate. mRNA for IL-2, IL-12R{beta}2, IL-4, IL-10, IL-12R{beta}2, and FasL were not detected at this time in the PVG/C6- rats (Fig. 6GoB). The level of IFN-{gamma} and Fas mRNA was also significantly lower in the PVG/C6- rats compared with PVG/C. The results correlate with the clinical status of the two groups, suggesting the level of cytokine mRNA expression in the spinal cord was consistent with the level of mononuclear infiltration rather than the differences in the expression of the Th subsets.



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  		FIGURE 6. Representative result of semiquantitative RT-PCR of cervical spinal cord samples. One of three sample in each group is shown, because results were similar within each group. A, RT-PCR products detected at day 14 postimmunization. There were no detectable differences in mRNA expression for IL-4, IFN-{gamma}, TNF-{alpha}, TGF-{beta}, Fas, and FasL between the PVG/C (n = 3) and PVG/C6- (n = 3) rats. There were greater levels of mRNA for IL-2, IL-12R{beta}2, and IL-10 in PVG/C compared with PVG/C6- rats. B, mRNA RT-PCR products detected at day 18 postimmunization showed limited or no detection of IL-2, IL-4, IL-10, IL-12R{beta}2, and FasL, and moderate levels of IFN-{gamma} and Fas were detected in PVG/C samples.

 
Examination of C3 and C9 deposition in spinal cords

The intensity of C3 deposition was similar between PVG/C6- and PVG/C rats. C9 deposition was detected in PVG/C but not in PVG/C6- samples, consistent with the lack of C6 and the formation ofthe MAC (Fig. 5GoA). Staining of C3 and C9 in PVG/C was greatest in the perivascular areas, where there were clusters of intense granular staining. Diffuse staining was found throughout the rest of the CNS. Normal PVG/C rat spinal cords had no staining for C3 and C9. EAE PVG/C6- rats had no staining for C9.

Examination of vascular adhesion molecules in spinal cords

ICAM-1 was present on infiltrating cells and vascular endothelium, and was less intense in PVG/C6- compared with PVG/C. There was a marked reduction of endothelial cell expression of ICAM-1 in PVG/C6- compared with PVG/C (Fig. 5GoA). There was P-selectin staining in the PVG/C on vascular endothelium, which was not seen in PVG/C6- (Fig. 5GoA) or normal brain tissue.

The expression of P-selectin and ICAM-1 mRNA was also examined by RT-PCR in three cervical cords from each group. As results within a group were similar, only one sample is shown in Fig. 6Go. The level of P-selectin and ICAM-1 were significantly less (2- and 4-fold, respectively, at day 14) in the PVG/C6- compared with the PVG/C group (Fig. 6GoA). At day 18 there was still a 2-fold greater level of ICAM-1 mRNA in PVG/C than in PVG/C6-. P-selectin was not detected in the PVG/C6- spinal cord at day 18 but was detected in a dilution of 1/10 in PVG/C (Fig. 6GoB). The RT-PCR findings correlated with the immunostaining findings.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As EAE is an archetypal T cell-mediated disease model, complement is thought to have a minor role in the pathogenesis of EAE. Recent studies on EAE in C3 knockout mice had contradictory results (17, 18). When active EAE is induced by immunization with a MOGp35–55 peptide, the C3 knockout in C57BL/6 mice develops less severe disease compared with the wild type in one study (17), but this was not observed in another study using B6 x 129/F1 C3 knockout (18). CVF, which depletes C3, can also reduce the severity of active EAE (5, 31). These studies do not differentiate between the lack of C3 for effective Ag presentation or reduced MAC formation. Our results with C6-deficient rats support a role for MAC activation in the development of EAE. C6 is not known to have any biological role apart from being required for assembly of the MAC; thus, the effect demonstrated in this study most likely relates to MAC itself (27).

This study demonstrated that PVG/C6- rats developed a milder form of active EAE with reduced inflammation in spinal cord compared with wild-type PVG rats (PVG/C). Reduced clinical disease in PVG/C6- rats was associated with both reduced mononuclear cell infiltration and levels of cytokine mRNA for activated mononuclear cells including Th1 and Th2 cytotoxic T cells and activated macrophages. This reduced macrophage and T cell infiltrate was similar to that reported in factor B and C3 knockout mice (17). Analysis of the node draining the site of immunization showed similar enlargement in PVG/C6-. Further studies of the T cell response in PVG/C6- was comparable to that of PVG/C as in the nodes, where there were equivalent levels of mRNA for Th1 cytokines IL-2, IFN-{gamma}, TNF-{alpha}, and the Th1 cell marker IL-12R{beta}2, as well as Th2 cytokines IL-4 and IL-10. The titer of the anti-MBP Ab response and complement-fixing isotype was not different in PVG/C and PVG/C6-, except there was a reduction of IgG1 in PVG/C6- rats. IgG1 has a low capacity to fix complement (39), and this isotype switch is driven by Th2 cytokines such as IL-4 (40). We did not demonstrate reduced Th2 cytokine induction in PVG/C6-; thus, the mechanism of this effect remained unexplained. Complement-fixing IgG isotypes were similar to PVG/C rats, which suggested the failure to deposit C9 was not due to lack of complement-fixing anti-MBP Abs. In a recent study, up-regulation of IgG1 production is associated with hypersensitivity to proteolipid protein peptide 139–151-induced EAE (41), but how such an effect may be related to our findings was not resolved. Taken together, our results suggest that the terminal complement components may play an important role in the effector phase of actively induced EAE but not in the induction of the immune response to MBP. Direct MAC-induced oligodendrocyte injury, either through lytic or sublytic pathways, is the possible mechanism of MAC-related injury to the CNS that may be impaired in PVG/C6- rats.

The reduced mononuclear cell infiltration in the PVG/C6- in our study did not appear to be related to impaired T cell activation, but rather may reflect the reduced recruitment of mononuclear cells to the CNS due to lower expression of adhesion molecules on vascular endothelium. The vascular adhesion molecules P-selectin and ICAM-1 can be up-regulated by sublytic activity of the MAC (42, 43), which activate endothelial cells (44, 45). The reduced ICAM-1 expression in PVG/C6- rats was similar to that observed in C3 knockout (17). These findings suggest MAC-induced breakdown of the BBB may be another mechanism by which EAE is exacerbated. This contribution of MAC by induction of ICAM-1 and P-selectin expression on vascular endothelium-promoted cell migration would explain the lesser inflammatory infiltrate in PVG/C6- rats. The reduced infiltrate may also account for the reduced severity of EAE in PVG/C6- rats.

Although it has been suggested that MAC may have a role in the expression of EAE (5, 31), the relative contribution of MAC compared with earlier complement components is unclear (24, 46). The overall importance of complement in CNS inflammation and regulation is supported by evidence that immunocompetent cells such as astrocytes, oligodendrocytes, and microglia can fully activate and synthesize complement components (47, 48, 49). Complement activation in the CNS can also occur even when the BBB remains intact (48, 50, 51). These findings suggest that local synthesis of complement may exacerbate neuronal damage independent of complement synthesis in the liver. The ability of brain cells to produce complement demonstrates the importance of complement as part of the innate immune regulatory mechanism in the CNS (52).

The disparity in the number of circulating leukocytes, particularly neutrophils between the PVG/C and PVG/C6- rats, is not readily explained, unless activation of MAC in the CNS induces leukocytosis. Studies of cultured endothelium have shown that complement fixation on vascular walls can cause integrin-dependent neutrophil adhesion (53). The MAC itself can induce neutrophil adhesion by augmentation of TNF-{alpha}-induced E-selectin and ICAM-1 (43). Studies of the role of complement in xenograft rejection have shown that complement and neutrophil activation may play a synergistic role in the pathogenesis of rejection (54), and this may involve the up-regulation of adhesion molecules on neutrophils (55, 56). Normally, endothelium is protected from complement activation, and hence inflammation, but vascular endothelium can become susceptible to complement activation following alteration of the morphology of the endothelial monolayer by cytokines (57). Whether the persistent leukocytosis in PVG/C rat was the cause or effect of the greater severity of EAE was not resolved by our studies.

The reduced severity of disease in MOG peptide-induced EAE in C3 and factor B knockout may also be in part due to lack of activation of C6 and formation of MAC. It is likely that there was normal C3a and C5a activation in PVG/C6- rats and that these inflammatory factors alone were insufficient to promote a full inflammatory response. Furthermore, normal C3 activation in PVG/C6- would have allowed promotion of the normal T cell response, as C3 is important in activating adaptive immune responses to protein Ags.

The mechanism leading to the development of CNS injury in EAE is complex and it is thus unlikely that a single complement component would effect the normal course of EAE (58). Similarly, the mechanisms by which MAC may contribute include MAC-mediated injury induced by complement-fixing Ig to MBP, as well as a reduced infiltration of T cell and mononuclear cells due to reduced expression of P-selectin and ICAM-1 on vascular endothelium. MAC either directly or indirectly also induce leukocytosis. Alone or together, these mechanisms may be the cause of the reduced severity of active EAE in PVG/C6- compared with PVG/C rats.


   Acknowledgments
 
We appreciate the work of Moheb Boutros in animal care and Dr. Mark Penny in characterizing the PVG/C6- at Liverpool Hospital. We thank Dr. X. Y. He for helpful advice and the staff in the Department of Haematology for their help on blood cell counts.


   Footnotes
 
1 This work was supported by the National Health and Medical Research Council of Australia and the Ingham Health Research Foundation of the South Western Sydney Area. Back

2 G.T.T. and S.J.H. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Suzanne J. Hodgkinson, Department of Medicine, University of New South Wales, Liverpool Hospital, Locked Bag 7103, Liverpool BC, NSW 1871, Australia. E-mail address: s.hodgkinson{at}unsw.edu.au Back

4 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; CVF, cobra venom factor; FasL, Fas ligand; MAC, membrane attack complex; MOG, myelin oligodendrocyte glycoprotein; BBB, blood brain barrier; MBP, myelin basic protein; WBC, white blood cells. Back

Received for publication June 8, 2001. Accepted for publication February 7, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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