Schwann cell extracellular matrix protein production is modulated by Mycobacterium leprae and macrophage secretory products

Journal of Neurological Sciences 151 (1997) 13–22

Schwann cell extracellular matrix protein production is modulated byMycobacterium leprae and macrophage secretory products

*Neeta Singh, Tannaz J. Birdi , Sushila Chandrashekar, Noshir H. Antia

The Foundation for Medical Research, 84-A, R.G. Thadani Marg, Worli, Bombay 400 018, India

Received 23 October 1996; revised 10 March 1997; accepted 25 March 1997

Abstract

Extracellular matrix (ECM) protein deposition is an important feature of leprous nerves, where Schwann cells (SCs) and macrophagesare the main hosts for Mycobacterium leprae. Since, SCs are involved in the synthesis of ECM proteins and its production is regulated bymacrophage secretory factors, the present study aimed to determine in vitro, the effect of M. leprae infection and macrophage secretoryproducts on secretion of ECM proteins by SCs in two strains of mice, Swiss White (SW) and C57BL/6, that are known to differ in theirnerve pathology and macrophage functions in response to infection. Following six days of M. leprae infection, SCs from SW mice

14responded with increased secretion of C-leucine radiolabelled proteins and a concomitant increase in laminin and collagens type I, IIIand IV, as determined by enzyme-linked immunosorbent assay. In contrast infected C57BL/6 SCs responded with decreased secretion oftotal proteins and fibronectin. Exposure of SCs to macrophage conditioned medium resulted in decreased ECM protein secretion in bothstrains of mice. This decrease was a function of protein breakdown by macrophage derived proteases and also active regulation bymacrophage secreted cytokines. A similar effect of M. leprae and macrophage secretory products on SC metabolism in leprous nerveswould have major ramifications on damage and repair activities. In addition ECM proteins would also influence the composition of theinfiltrating cell population in lepromatous and tuberculoid nerves. 1997 Elsevier Science B.V.

Keywords: Schwann cells; Mycobacterium leprae; Macrophages; Laminin; Collagens; Fibronectin

1. Introduction Previous studies have shown that the ability of SCs toensheath and myelinate axons depends on deposition of the

Collagens, laminin and fibronectin form the bulk of the BL and absence of any of the BL components can lead toextracellular matrix (ECM) proteins found in the peripher- aberrant SC–axon associations (Eldridge et al., 1989;al nerve. Collagens type I and type III are localised in the Obremski et al., 1993) resulting in severe neurologicalinterstitial spaces of the epineurium, perineurium and the manifestations (Okada et al., 1980; Cornbrooks et al.,endoneurium. Collagen types IV and V, laminin and 1983). Besides this, all the three ECM proteins promotefibronectin are constituents of the basal lamina (BL) that neurite outgrowth and therefore play an important role insurrounds each Schwann cell (SC)–axon unit and the peripheral nerve regeneration (Manthorpe et al., 1983;perineurial layers (Shellswell et al., 1979; Sanes, 1982). Adler et al., 1985). On the other hand excessive depositionOver the last two decades sufficient evidence has emerged of ECM proteins, mainly collagens and fibronectin, leadsthat show SCs to be one of the major contributors of neural to tissue scarring and fibrosis in chronic progressiveECM proteins (Bunge et al., 1980; Carey et al., 1983). conditions that in time obliterate the tissue structure

(Haralson, 1993).* Macrophages, which have been shown to be activelyCorresponding author. Tel.: 191 22 4934989/191 22 4932876; Fax:

191 22 2662735. involved in assisting SCs in peripheral nerve repair (Perry

0022-510X/97/$17.00 1997 Elsevier Science B.V. All rights reservedPII S0022-510X( 97 )00105-6

14 N. Singh et al. / Journal of Neurological Sciences 151 (1997) 13 –22

and Brown, 1992) are also involved in connective tissue 2. Materials and methodremodelling both in repair and in pathological conditions.This is due to their capacity to produce proteases, in- 2.1. Micehibitors to these proteases, cytokines and prostaglandinsthat regulate ECM metabolism (Cury et al., 1988; Border SW and C57BL/6 strains of mice were used for theand Rouslahti, 1992; Zahner et al., 1994; Butt et al., studies. SCs were obtained from 1–2 day postnatal mice1995). and 3–4 month old animals were the source for peritoneal

M. leprae has a unique affinity for SCs (Job, 1979; macrophages.Mukherjee and Antia, 1985) and since SCs are im-portant in nerve regeneration, any alteration in their 2.2. Cell culture preparationfunctions following infection with M. leprae wouldhamper nerve repair in leprous nerves. Even though 2.2.1. Dissociated SC culture (DSC)evidence for regeneration exists in the early leprous Cultures were essentially obtained by a modified methodnerve (Shetty et al., 1988), studies have shown that of Brockes et al. (1979). Briefly, sciatic and brachialinfection of SCs by M. leprae in vitro results in plexus nerves were collected under aseptic conditions,decreased proliferation (Mukherjee et al., 1989), expres- chopped finely and treated with a 1:1 solution of 0.25%sion of cell surface molecules NgCAM (Singh et al., trypsin and 0.05% collagenase (Sigma, USA). They were1996) and nerve growth factor receptor, p75 (Singh et dissociated by triturating through a 23G needle and platedal., 1997), which are functions associated with nerve on culture dishes in Dulbecco’s Modified Eagles Mediumregeneration. In addition macrophages initially form the (DMEM) (Gibco, USA) supplemented with 10% fetal calfbulk of the infiltrating cells in nerves of both lepromat- serum (FCS) (Gibco) and antibiotics, penicillin and strep-ous and tuberculoid patients and in vitro have been tomycin. For determination of proteins secreted into thereported to enhance the expression of NgCAM on the culture medium, the cells were plated at a density ofSC surface (Singh et al., 1996). 300 000 cell /55 mm Petri dish and for cellular emzyme-

In leprous nerves indications of affected ECM metabo- linked immunosorbent assays (ELISAs) at a density oflism comes from electron microscopic observations of 20 000 cells /well of a 96 well plate. The cultures werecollagen pockets around affected nerve fibers (Antia et al., incubated at 378C in an atmosphere of 5% CO . After 482

251980; Shetty et al., 1980a,b) in the early stages of damage h, the cells were treated with 10 M cytosine arabinosideand constant fluctuation in the BL structure at different (Sigma) for 48 h to minimise the fibroblast population.stages of the disease. Besides this, fibrosis is one of the Cultures thus obtained were 93% pure for SCs, as de-major factors that contributes towards the irreversibility of termined by S100 immunofluoresence staining (Brockes etnerve damage in leprosy (Job, 1971, 1973). It is not known al., 1979), while the remaining percentage consisted of awhether these pathological features are a reflection of mixture of fibroblasts and neuroepithelial cells. Five to sixaltered ECM metabolism of SCs following infection with day old cultures were used for infection with M. leprae.M. leprae or exposure to macrophages and their secretoryproducts. Therefore the aim of this study was to assess, in 2.2.2. Peritoneal macrophage culturevitro, secretion of ECM proteins viz. laminin, fibronectin Macrophages were obtained by irrigating the mouseand collagen types I, III, IV and V by M. leprae infected peritoneal cavity with Minimum Essential MediumSCs and to determine if secretory products of M. leprae (MEM) (Gibco). The cells thus obtained were plated on 55infected macrophages are capable of modulating ECM mm Petri dishes (5 million cells /plate) in medium consist-production by SCs. ing of MEM supplemented with 10% FCS and penicillin.

All the studies were compared in cells derived from two After 24 h the cultures were washed free of nonadherentstrains of mice, Swiss White (SW) and C57BL/6 as they cells and maintained for an additional five to six daysdiffer in their response to M. leprae infection. Firstly, in before infection with M. leprae or activation withspite of comparable M. leprae growth in the foot pad and lipopolysaccharide (LPS).similar involvement of unmyelinated fibers of sciaticnerves in the early stages of infection, nerve damage 2.3. Source of M. leprae and infection of cell culturesprogresses to extensive demyelination only in SW mice(Birdi et al., 1995). Secondly SW peritoneal macrophages M. leprae was derived from liver and spleen biopsies oflike those from lepromatous patients are rendered defective experimentally infected armadillos (supplied by Dr E.on infection with M. leprae, while macrophage functions Storrs, Florida Institute of Technology). The tissues werein C57BL/6 mice, like tuberculoid patients and normal homogenised in DMEM, subjected to differential centrifu-individuals remain unaffected on infection with M. leprae gation to minimize tissue contamination, stored at 48C and(Birdi et al., 1983; Salgame et al., 1983; Birdi and Antia, used within a week.

61989). SC and macrophage cultures, were infected with 10 /ml

N. Singh et al. / Journal of Neurological Sciences 151 (1997) 13 –22 15

viable or heat-killed (autoclaved for 20 min at 1208C and bated for 3 h at 378C. Monoclonal antibody to laminin15 lbs) M. leprae for 24 h after which extracellular bacilli (Serotec, code MCA 224) was used at a dilution of 1:1000were washed off. and incubated for 1 h at 378C and to fibronectin (Seralab,

code. MAS 37C) was used at a dilution of 1:2000 and2.4. Activation of macrophages with LPS incubated overnight at 48C. HRP labelled secondary anti-

bodies (Dakopatts, Denmark) were used at a dilution ofLPS (2 mg/ml) (Sigma) was added to macrophage 1:2000 for collagen and 1:1000 for laminin and fibro-

cultures for 2 h after which the excess LPS was washed nectin. The protocol followed for the ELISAs is indicatedoff. Cultures were maintained for a further 24 h in MEM in Fig. 1.supplemented with 10% FCS and penicillin followingwhich the conditioned medium was collected and used. 2.8. Standard collagen type I levels in presence of

collagenase /macrophage conditioned medium2.5. Addition of macrophage conditioned medium to SCcultures A 50 ml volume of collagen type I (1 mg/ml in DMEM)

was added to equal volume of collagenase (5 mg/ml inConditioned medium from uninfected, 3 day post-infec- DMEM) or macrophage conditioned medium in a 96 well

ted and LPS activated macrophage cultures were cen- plate and incubated for 72 h at 378C. Following this, 50 mltrifuged at 4000 rpm to remove extracellular bacilli and of the reaction mixture was coated in a 96 well Nunccellular debris. They were added immediately to SC Maxisorp plate, for 72 h at 48C. Levels of collagen type Icultures with an equal volume of DMEM supplemented were determined as described in Fig. 1.with 10% FCS and antibiotics and maintained for 72 h. Forstudies that required SCs to be maintained with the 2.9. Statistical analysisconditioned media for longer durations, medium from afresh set of macrophage cultures, treated as described Statistical significance was determined by the Student’sabove, was added every 72 h. unpaired t-test.

2.6. Estimation of secretory proteins

SC cultures in 55 mm Petri dishes were pulsed with14C-leucine (5 mCi/plate) (Amersham, Denmark, Sp.activity 11.7 GBq/mmol) in 2.5 ml of leucine free DMEM

14(Sigma) supplemented with 10% FCS and antibiotics. C-leucine was added to 3 day post-infected and corre-sponding uninfected SC cultures. Thereafter spent mediumwas collected every 72 h and cultures re-pulsed with fresh

14leucine free medium containing C-leucine for the nexttwelve days. In some plates along with the initial additionof radiolabel, cycloheximide at concentrations 5, 10 and 15mg/ml was also added. Proteins in 2 ml of the collectedconditioned medium were precipitated by treating twicewith 20% trichloroacetic acid. The protein precipitate wasdissolved in 1 N NaOH, added to 10 ml of Brays fluid andradioactivity determined in a scintillation counter. Part ofthe culture supernatant was used to determine levels ofsecreted collagens and fibronectin.

2.7. ELISA for collagens type I, III, IV and V,fibronectin and laminin

Conditioned medium from SC cultures in 55 mm plateswas assayed for collagens and fibronectin. For laminin, a

Fig. 1. ELISA protocol for the determination of collagens, fibronectin andcellular ELISA on SC cultures in 96 well plates waslaminin. Dilutions of primary and secondary antibodies and incubation

performed. Primary antibody to collagens type I, III, IV or temperature and duration used as mentioned in Section 2.7. Abbrevia-V (Sera-lab, code nos. 1310, 1330, 1340 and 1350, tions: PBS, phosphate buffer saline; BSA, bovine serum albumin; HRP,respectively) was used at a dilution of 1:1000 and incu- horseradish peroxidase.


3. Results

3.1. SC functions in response to M. leprae infection

3.1.1. Total protein secretionSW and C57BL/6 SCs responded differently to in-

fection with M. leprae. In SW mice significantly higherlevels of radiolabelled proteins were detected in culturesupernatants (P,0.005), while in C57BL/6 DSCs a de-crease was observed (P,0.001). In both strains, the

14maximum difference in the levels of C-leucine labelledproteins secreted by infected versus uninfected cells wasobserved at six days post-infection (Fig. 2a). Therefore insubsequent experiments individual ECM proteins wereestimated in culture supernatants collected from 6 daypost-infected SCs.

As the age of SC cultures increased, the amount ofradiolabelled proteins secreted by the cells decreasedsteadily, though the trend of protein secretion of infectedcells versus uninfected cells remained unchanged until thetwentieth day post-infection in both strains (Fig. 2a).

Secretion of radiolabelled proteins was also studied inthe presence of cycloheximide. A dose dependent decreasewas observed in 6 day post-infected SCs from both strainsof mice (Fig. 2b).

3.1.2. Cell surface lamininFollowing infection with viable M. leprae for six days,

SCs from SW mice expressed increased levels of laminin(P,0.005) on their cell surface while levels remainedunchanged in C57BL/6 mice. Infection of SCs with heat-killed M. leprae had no effect on the levels of cell surfacelaminin expression in both strains (Table 1).

3.1.3. Collagen secretionSecretion of collagens types I, III and IV were increased

(P,0.05) in SW SCs following infection with M. lepraefor six days. In comparison, C57BL/6 SCs showed nochange in their collagen secretion pattern in response toinfection. In the two strains, infection of SCs with heat-killed M. leprae for a similar time period did not have anyeffect on secretion levels of any of the collagen typesstudied (Table 1).

3.1.4. Fibronectin secretionFollowing infection for six days, secretion of fibronectin

Fig. 2. (a) Secretion of proteins by uninfected (———d———) and M.was decreased (P,0.01) in SCs from both strains of miceleprae infected (– – ? – –) SCs from SW and C57Bl /6 mice. Trichloro-(Table 1). Infection with heat-killed M. leprae had no 14acetic acid precipitates of C-leucine labelled protein in culture super-

effect on the secreted levels of fibronectin in the two natants, collected at three day intervals, were added to Brays fluid andstrains. counts were determined in a scintillation counter. Values are expressed as

average cpm counts6SD of three individual experiments. (b) Effect ofcycloheximide on the secretion of proteins by uninfected SCs (———3.2. SC functions in response to macrophage secretoryd———) and SCs infected with M. leprae for 6 days (– – ? – –), fromproducts. 14SW and C57Bl /6 mice. DSC cultures were pulsed with C-leucine, withand without cycloheximide, for 72 h following which counts of radio-

3.2.1. Total protein secretion labelled proteins in culture supernatants were determined. Values givenUninfected SCs from both strains responded with a are mean cpm counts of two individual experiments.


Table 1Effect of M. leprae infection on Schwann cell ECM proteins

Laminin Fibronectin Coll. I Coll. III Coll. IV Coll. V

(a) SWUninfected 1.09760.07 1.25860.09 0.96560.02 0.78560.09 0.72260.03 0.91160.02

a b c c c1M. leprae 1.70160.02 0.89560.02 1.30660.01 1.09960.02 1.16260.04 0.89160.091heat-killed M. leprae 1.12360.02 1.12360.08 0.89360.05 0.67960.007 0.78760.03 0.90160.07

(b) C57BL/6Uninfected 0.9360.07 1.2160.015 1.26760.02 0.79260.01 0.69860.1 0.95760.08

b1M. leprae 0.8860.03 0.61360.09 1.06260.09 0.7160.07 0.7360.02 1.01560.011heat-killed M. leprae 0.9260.08 1.17260.09 1.09860.02 0.76160.1 0.62160.07 0.89660.05

Cellular laminin levels and secreted levels of fibronectin and collagens type I, III, IV and V, as determined by indirect ELISA, of uninfected Schwann cellswere compared with Schwann cells infected for six days with viable or heat-killed M. leprae in (a) SW and (b) C57BL/6 strains of mice. Values given aremean of optical density (OD) at 492 nm6SD of three independent experiments each performed in triplicate. Difference in OD significant in comparison to

a b cuninfected SCs: P,0.005; P,0.01; P,0.05.

decrease (P,0.001) in secretion of radiolabelled proteins in culture supernatants of uninfected SW SCs, with theon exposure to conditioned medium from uninfected, exceptions being collagen type IV, levels of which re-infected and LPS activated macrophage cultures. In con- mained unaltered, and collagen type I, levels of whichtrast, opposing effects of macrophage conditioned medium were decreased only in the presence of M. leprae infectedwas observed on M. leprae infected SCs in the two strains or LPS activated macrophage conditioned medium. Inter-of mice. In SW SCs, where M. leprae infection had estingly in infected SW SC culture supernatants, the levelsresulted in elevated levels of proteins secretion, a decreasewas observed following exposure to macrophage secretoryproducts (P,0.01). In C57BL/6 DSCs, where followinginfection a depression in protein secretion was observed,exposure to macrophage secretory products resulted in anenhancement (P,0.02) in the levels of secreted proteins(Fig. 3).

3.2.2. Cell surface lamininLaminin levels were regulated differentially in the two

strains. Uninfected and M. leprae infected C57BL/6 SCs,which had shown no change in their cellular laminin levelson infection with M. leprae for six days, showed ageneralised decrease in laminin levels on exposure tomacrophage conditioned medium.

Uninfected SW SCs showed significant enhancement(P,0.005) in laminin levels in the presence of uninfectedand infected macrophage conditioned medium while adecrease (P,0.05) was observed in the presence ofconditioned medium from LPS activated macrophages.Infected SW SCs, which had responded with increasedcellular levels of laminin following infection with M.leprae for six days, showed a decrease (P,0.02) inlaminin levels following exposure to macrophage con-ditioned medium (Table 3).

3.2.3. Collagen secretion 14Fig. 3. C-leucine labelled proteins secreted by uninfected SCs (h) andDecreased levels of collagens types I, III (Table 2), and SCs infected with M. leprae for six days ( ) on exposure to macrophage

V (data not given) were detected in supernatants of both conditioned medium as compared to control SCs unexposed to con-ditioned medium. Uninfected SC and SCs infected with M. leprae foruninfected and M. leprae infected C57BL/6 SCs exposed

14three days were pulsed for 72 h with C-leucine along with conditionedto macrophage conditioned medium. The only exceptionmedium from macrophages that were uninfected (MoU), infected for

was collagen type IV (Table 3), levels of which remained three days with M. leprae (MoI) or LPS activated (MoLPS). Values givenunchanged following exposure to the conditioned media. are mean cpm counts6SD of three independent experiments, eachSimilarly levels of collagen types III and V were decreased performed in triplicate.


Table 2Effect of macrophage secretory products on secretion of collagens type I, III and IV by Schwann cells from SW and C57BL/6 mouse strains

Schwann cell1macrophage CM Collagen Type I Collagen type III Collagen type IV

SW C57Bl /6 SW C57Bl /6 SW C57Bl /6

SCU 0.98160.03 1.29060.02 0.78460.07 0.78260.02 0.75360.02 0.70760.03SCI 1.31060.02 1.06260.10 0.99660.03 0.73060.05 1.15460.03 0.74360.02SCU1MoU 0.84760.04 0.73460.02 0.51760.04 0.49960.06 0.61860.02 0.62160.05SCI1MoU 0.91960.01 0.72160.01 0.44660.06 0.39960.07 0.51260.06 0.41060.02SCU1MoI 0.67260.02 0.61660.02 0.49460.01 0.46260.02 0.69760.01 0.69960.04SCI1MoI 0.74060.02 0.89260.01 0.46660.03 0.36260.09 0.61960.03 0.40060.02SCU1MoLPS 0.71660.02 0.61760.02 0.40260.07 0.49060.07 0.69060.01 0.49460.02SCI1MoLPS 0.78360.02 0.71560.01 0.43160.03 0.41360.02 0.61260.02 0.46160.03

Secreted levels of collagens type I, III or IV, as determined by indirect ELISA, of uninfected Schwann cells (SCU) and Schwann cells infected with M.leprae for six days (SCI) were compared with Schwann cells exposed for 72 h to conditioned medium (CM) from macrophage cultures that wereuninfected (MoU), infected with M. leprae for three days (MoI) or activated with LPS (MoLPS). Values given are mean of optical density (OD) at 492nm6SD of three independent experiments each performed in triplicate.

of the four collagen types, especially of type I, III and IV macrophage conditioned medium without aprotinin (datawhose levels had increased following M. leprae infection, not given).decreased on incubation with macrophage conditionedmedium (Table 2). 3.2.4. Fibronectin secretion

Effect of macrophage conditioned medium on standard A generalised decrease was observed in the culturecollagen type I (1 mg/ml) was also studied to determine supernatant levels of fibronectin in uninfected SCs fromwhether the decrease was due to secretion of collagenase both the strains on incubation with macrophage con-by the macrophages. A decrease in collagen was observed ditioned medium. Fibronectin levels of M. leprae infected(P,0.005) following exposure to conditioned medium SCs from the two strains, which had already decreasedfrom uninfected or M. leprae infected macrophage cultures following infection, were reduced further (P,0.02) fol-(Fig. 4). In the same experiment inclusion of a protease lowing exposure to the conditioned media (Table 3).inhibitor, 25 mg/ml aprotinin (Sigma) resulted in protec-tion against the action of macrophage conditioned mediumas indicated by a significant increase (P,0.05) in immuno- 4. Discussionreactivity to collagen type I (Fig. 4). Both these observa-tions indicate collagen breakdown by macrophage derived The production of ECM components is a major functionproteases. However, when secretion of the collagen by SCs of SCs during nerve repair. In this study we have reportedin response to macrophage conditioned medium containing that both M. leprae infection per se and secretory productsaprotinin was estimated, no change in levels of the of uninfected and M. leprae infected macrophages modu-collagen was observed as compared to cells exposed to late this aspect of SC function, thereby implicating altered

Table 3Effect of macrophage secretory products on Schwann cell fibronectin secretion and cellular laminin in SW and C57Bl /6 mouse strains

Schwann cell1 macrophage CM Fibronectin Laminin

SW C57Bl /6 SW C57Bl /6

SCU 1.21160.10 1.19860.03 1.11260.09 0.99160.09SCI 0.91060.03 0.69960.08 1.69160.03 0.84260.05SCU1MoU 0.78060.05 0.21960.07 1.45060.07 0.61060.08SCI1MoU 0.56160.06 0.14560.05 1.35060.07 0.62060.07SCU1MoI 0.54660.07 0.23760.07 1.86060.02 0.54760.04SCI1MoI 0.52460.02 0.19660.03 0.80760.11 0.56060.05SCU1MoLPS 0.45360.05 0.16160.09 0.77360.07 0.55060.04SCI1MoLPS 0.42460.06 0.13860.03 0.62060.01 0.58160.04

Levels of secreted fibronectin and cellular laminin, as determined by indirect ELISA, in uninfected Schwann cells (SCU) and Schwann cells infected withM. leprae for six days (SCI) were compared with Schwann cells exposed for 72 hrs to conditioned medium (CM) from macrophage cultures that wereuninfected (MoU), infected with M. leprae for three days (MoI) or activated with LPS (MoLPS). Values given are mean of optical density (OD) at 492nm6SD of three independent experiments each performed in triplicate.


4.2. Production of BL and interstitial ECM proteins byM. leprae infected SCs

In the C57BL/6 strain, the levels of laminin andcollagen types IV and V were not affected. Consequentlythe deposition of BL by SCs would most probably not beadversely affected. In infected SW SCs, levels of the BLcomponents were altered substantially with increasedlevels of collagen type IV and laminin. Even though thisresponse appears positive, such a response in vivo wouldnot necessarily result in the deposition of a functional BL.Addition of BL components to dorsal root ganglioncultures have been shown to result in exaggerated enshea-thing structures of axons which resemble unmyelinatedmorphology found in some peripheral neuropathies (Ob-remski and Bunge, 1995). In addition, sciatic nerves of SWmice inoculated with M. leprae in the foot pad show thepresence of polyaxonal myelination (Antia and Shetty,1984) which might reflect the inability of SCs in a leprous

Fig. 4. Effect of peritoneal macrophage conditioned medium on collagennerve to assemble proper BL.type I. Level of 1 mg/ml collagen (h) was determined following

The secretion of interstitial collagens type I and III wasexposure for 72 h, with or without aprotinin (Ap), to (a) collagenase ( ),increased in SW SCs following infection while levels(b) conditioned medium from uninfected macrophages ( ) or (c)

conditioned medium from macrophages infected with M. leprae for three remained unchanged in the C57BL/6 strain. Thoughdays (j). Values expressed as mean optical density (OD) at 492 nm6SD collagenization is a feature of nerves from patients acrossof one experiment performed in triplicate.

the spectrum, it is not known which cells or what otherfactors contribute to the fibrosis. These results indicate that

ECM protein metabolism of SCs as one of the factors at least in SW mice and presumably lepromatous patientscontributing to leprous neuropathy. increased production of the interstitial collagens I and III

by M. leprae infected SCs would contribute substantiallyto collagenization in these nerves.

4.1. Protein secretion by SCs in response to M. lepraeinfection 4.3. Total proteins, ECM proteins and macrophage

secretory productsDecrease in protein secretion by SCs in the presence of

cycloheximide indicate that the radiolabelled proteins in Response of SCs to macrophage secretory proteins wasthe culture supernatants were secreted by metabolically largely not dictated by M. leprae infection of macro-active SCs. There appears to be active modulation, though phages, as the action of macrophage secretory products ondifferently in SW and C57BL/6 mice, of SC protein ECM secretion by SCs was similar regardless of whethersecretion as a consequence of M. leprae infection. Further they were uninfected, infected or activated with LPS. Onthis modulation was not just a consequence of M. leprae the whole uninfected and M. leprae infected SCs fromuptake as indicated by unaltered levels following infection both the strains responded with a general decrease in thewith heat-killed bacilli. Alteration in protein synthesis levels of ECM proteins, with the exception being lamininfollowing infection with viable M. leprae is a well levels of uninfected SW SCs which increased on exposuredocumented observation in macrophages from lepromatous to macrophage secretory products. Macrophages constitu-patients and SW mice (Birdi et al., 1979; Kurup and tively secrete cytokines like fibroblast growth factorMahadevan, 1982), where a depression in protein synthesis (FGF), transforming growth factor (TGF-b), platelet de-has been observed. In these studies macrophages from rived growth factor (PDGF), which are considered to betuberculoid patients, C57BL/6 mice and normal individ- fibrogenic in nature as they either enhance cell prolifer-uals did not show any changes in protein synthesis levels. ation and secretion of ECM proteins or induce expressionIn the present study, response of SCs to M. leprae of ECM proteins at the genetic level (Kovacs, 1991). Theyinfection reiterates the ability of M. leprae to induce also secrete cytokines that have been shown to be suppres-alterations in the protein secretion of its host cells. Though sive in their actions on ECM metabolism and primarilythe M. leprae induced modulation of SC protein secretion include prostaglandins and tumor necrosis factor (TNF-a)is not analogous to that observed in macrophages from the (Mauviel et al., 1988; Zahner et al., 1994). In addition,two strains, the effect is nevertheless distinct in the two macrophages secrete a repertoire of protein degradingstrains. enzymes which includes matrix metalloproteinases that


degrade interstitial collagens type I and III, stromeolysin infection per se to patient nerve pathology will not bethat act on laminin and fibronectin and a 72 kDa collagen- apparent, but may contribute to the composition of thease that cleaves basement membrane collagens type IV and infiltrating cell population, with lymphocytes predominat-V (Welgus et al., 1985; Cury et al., 1988; Hibbs et al., ing in the tuberculoid nerve and macrophages prevalent in1987). In the present study, in addition to the suppressive the lepromatous (Pearson and Ross, 1975).actions of cytokines, a major contribution of macrophage Macrophages from the two strains, which otherwisederived proteases is also indicated in the repressive actions differ in their responses to M. leprae, modulated SC ECMof macrophage conditioned medium on the ECM levels as: secretion in a similar manner. These macrophages could(a) exposure of standard collagen type I to macrophage contribute to nerve damage in three ways. Firstly, the ECMconditioned medium resulted in its breakdown; and (b) this proteins produced by SCs would be broken down by thedecrease was partly prevented by the inclusion of ap- proteases. Studies have shown that laminin, collagens androtinin, a protease inhibitor. Studies have also demon- fibronectin in their native or disintegrated forms arestrated that macrophage secretory products also induce the chemotactic to immunological cells (Malone et al., 1991;secretion of proteolytic enzymes in responsive cells (Bor- Odekon et al., 1991; Hershkoviz et al., 1993). Thereforeder and Rouslahti, 1992). It is likely that in the present the breakdown products of the ECM proteins would furtherstudy, the proteins secreted by SCs in culture would not amplify leprous nerve pathology as they would act asonly be exposed to macrophage derived proteases present signals for the additional recruitment of inflammatoryin the conditioned medium, but also to self secreted cells. Secondly, on coming in contact with collagen andproteolytic enzymes induced by macrophage secretory their breakdown products, the macrophages would beproducts. However, in culture a similar recovery in col- induced to produce reactive oxygen intermediates (ROI)lagen type I levels in SW and C57BL/6 SCs was not (Laskin et al., 1994), which in addition to killing theobserved on exposure to macrophage conditioned medium bacilli, would also cause nonspecific bystander damage toin the presence of aprotinin, indicating that the suppressive the nerve. In SW mice higher levels of collagen would notaction of macrophage conditioned medium on ECM pro- necessarily result in increased ROI production as M. lepraetein production cannot be entirely attributed to proteases. infected macrophages from this strain are recalcitrant toTherefore the effect of macrophage secretory products on further activation (Watson et al., 1984; Marolia andECM proteins in the present study appears to be a Mahadevan, 1988). Therefore the damage contributed bycombination of (a) active modulation by macrophage this mode would be higher in C57BL/6 mice. Finallysecreted cytokines and other factors and (b) macrophage ECM metabolism would also be subjected to negativeand self derived proteases. regulation by the macrophage suppressive factors resulting

in an overall decrease in ECM protein levels contributed4.4. Implication for leprous nerve pathology by SCs.

The negative effect of macrophage secretory factors onAlteration in ECM protein secretion of M. leprae ECM protein accumulation by SCs does not indicate a

infected SCs in the two strains, albeit to a lesser degree in major role for these cells in the collagenization observed inC57BL\6 mice, indicates an active role for SCs in the early the advanced leprous nerves. Therefore the role of otherleprous nerve pathology. The nerve in the early stages in neural cell constituents, mainly fibroblasts can be consid-the two strains and both lepromatous and tuberculoid ered in the fibrosis of leprous nerve. Fibroblasts are thepatients has a common nerve pathology and damage occurs major cells involved in wound healing and in pathologicalin the absence of infiltrating cells (Antia et al., 1980). conditions due to their high ECM protein metabolicTherefore it was hypothesised that the response of SCs to capacity which primarily includes the deposition of thethe presence of M. leprae would be similar. However in interstitial collagens type I and III (Butt et al., 1995).the present study a differential response was noted, with Parallel studies (unpublished observations) on the action ofC57BL/6 SCs being more resilient to changes in their M. leprae infection on neurofibroblast secretion of col-ECM protein secretion. This suggests that the divergent lagens have demonstrated a decrease in secretion ofresponse to infection may act as a trigger for the differing collagens type IV in SW strain and type I in C57BL/6sciatic nerve pathology in the later stages in the two strains stain. However, in the same study it was observed that(Birdi et al., 1995). C57BL/6 SCs with their relatively neurofibroblasts were comparably resistant to the suppres-unaltered ECM secretion following infection would aug- sive actions of macrophage secretory products, as onlyment regeneration and this indeed seems to be reflected in secretion of those collagens that were affected by M.the lower degree of nerve damage in the later stages as leprae infection per se were decreased in the presence ofcompared to SW mice. macrophage conditioned medium.

The murine model does not reflect the nerve pathology In summary, aberrant ECM protein metabolism of SCsseen in patient nerves in the later stages wherein heavy on infection with M. leprae and on exposure to macro-infiltration is associated with gross damage. Thus the phage secretory products can be implicated as an importantconsequence of dissimilar SC response to M. leprae factor in the neuropathogenesis of leprosy by directly


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Schwann cell extracellular matrix protein production is modulated by Mycobacterium leprae and macrophage secretory products