ORIGINAL ARTICLE

Inhibition of Mononuclear Cell Procoagulant Activity by Lipophosphoglycan of Leishmania donowuni Raffaele Del Prete, Maria Raffaella Buongiorno, Adriana Mosca, and Giuseppe Miragliotta

Background: Since fibrin formation is an expression of the response of the host to parasite spread, the lipo- phosphoglycan (LPG) of Leishmania donovani and its carbohydrate fragment (PG) were examined for their capacity to inhibit procoagulant activity (PCA) production by human mononuclear cells stimulated with Escherichia coli endotoxin in vitro.

Methods: the putative inhibitory effect of LPG and its PG fragment was evaluated on the basis of their in vitro capacity to prolong significantly the time required for coagulation induced by endotoxin-stimulated mononuclear cells.

Results: LPG exhibited the most inhibitory activity, whereas the carbohydrate domain was not effective. These results are in agreement with the notion that LPG (but not PG) has an inhibitory effect on protein kinase C activity which plays a key role in the production of PCA by human monocytes.

Conclusions: From a pathophysiological point of view, these data suggest the possibility that Leishmania avoids fibrin entrapment in the host through this inhibitory mechanism.

introduction

The protozoan parasite Leishmania donovani is the causative agent of the often life-threatening disease kala-azar. This organism survives in the host as an amastigote within circulating macrophages which are regularly infected after the bite of a blood-sucking sandfly vector (1). However, because of the relatively poor knowledge of the parasite’s cell biology, still fragmentary information is available about how the parasite avoids destruction in the host’s environment. It appears likely that the capacity of L. donovani to persist in both vector and host may be related to the presence of highly specialized molecules on the parasite’s cell surface. The recently identified major surface molecule

Raffaele Del Prete. M.D., Maria Raffaella Buongiorno, B.Sc., Adriana Mosca, BSc., and Giuseppe Miragliotta, M. D.: Institute of Medical Microbiology, University of Bari, Policlinico, Piazza G. Cesare, 1-70124 Bari, Italy.

Corresponding author and reprint requests: Giuseppe Miragliotta, M.D.: lstituto di Microbiologia Medica Policlinico, Piazza G. Cesare 1-70124 Bari, Italy.

Telephone: 39 80 5478486 Fax: 39 80 5478537

lipophosphoglycan (LPG) appears to be the candidate to play this important role in the host cell-parasite interaction (2, 3, 4). LPG is a heterogeneous glyco- conjugate containing, as its salient feature, a repeating phosphorylated disac-charide unit. The repeated units are attached to an unusual lysoalkylphosphatidylinositol lipid anchor via a phosphoheptasaccharide carbo- hydrate core ( 5 , 6, 7). O n the other hand, human mononuclear cells (i.e. monocytes and lymphocytes) produce a potent tissue factor-like procoagulant activity (PCA) in response to several stimuli, including the endotoxin of gram-negative bacteria (lipoplysaccharide or LPS) (8). In turn, this tissue factor-like procoagulant activity activates the clotting cascade through the extrinsic pathway of blood coagulation with formation of fibrin (9). Since fibrin formation is an expression of the response of the host, procoagulant activity pro- duction can be regarded as an important function of the immune response (10).

O n this basis, we have examined in our in vitro experiments the putative ability of L. donovani LPG and of its carbohydrate portion (PG) to inhibit the PCA production by LPS-stimulated human mononuclear cells and we suggest herein the importance of LPG for both survival and spread of the parasite.

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32 Journal of Cl inical Microbio logy and infection, Volume 1 Number 1

Materials and Methods

Lipopolysaccharide (LPS) endotoxin Escherichia coli LPS 0125:B8 was purchased from DIFCO Laboratories, Detroit Michigan, USA.

Lipophosphoglycan (LPGI LPG was isolated from stationary phase cultures of L. donovani promastigotes, as described (5). The entire carbohydrate portion termed “phosphoglycan” (PG) was obtained by treatment of LPG with phosphatidy- linositol-specific phospholipase C (1 1). Either LPG or PG were resuspended in sterile apyrogen saline and sonicated for 60 seconds before use. Suspensions were tested by Limulus amebocyte lysate assay (Whitaker Bioproducts, Walkersville, MD) in order to ascertain the absence of endotoxin.

Blood collection and preparation of mononuclear cells Blood was collected from healthy donors using trisodium citrate (0.015 M final concentration) as the anticoagulant. According to the method ofBoyum (12), mononuclear cells were isolated with the LeucosepTM device (Opopharma AG, Zurich, Switzerland) using Lymphoprep (Nycomed Fharma AS, Oslo, Norway) as a separation medium. The cells were washed three times with citrated Hanks’ balanced salt solution (HBSS) and then resuspended in HBSS without citrate at the desired concentration. Granulocyte contami- nation was less than 5% and the ratio of platelets to leukocytes was less than 1:l as determined by light microscopy. Twenty percent of these cells were identified as monocytes by cytochemical reactivity for a-naphtyl-acetate esterase (13). The suspensions of mononuclear cells were incubated with E. coli LPS (0.2pM final concentration), L. donovani LPG (0.2 pM final concentration), and E. coli LPS (0.2 pM final concentration) along with LPG at different concen- trations (0.4 pM, 1 pM, 2 pM, 4 pM). The negative control was represented by mononuclear cells in- cubated with apyrogen saline. In order to investigate the role played by the carbohydrate portion, another set of experiments was carried out using PG instead of LPG in the incubation mixtures.

Procoagulant assay After a 4-hour incubation at 37OC, PCA was evaluated by a one-stage plasma recalcification time. Citrated human plasma (pooled from at least three healthy donors) was used as a substrate. Clotting time was determined in duplicate in plastic tubes using a manual method on a mixture of 0.1 ml plasma, 0.1 ml cell suspension, 0.1 ml 0.025 M CaC12 (14).

When L. donovani -LPG or -PG was added to the E. coli LPS-mononuclear cells incubation mixtures, the percent PCA inhibition was derived from the assays described above using the formula (B - A)/A x 100 where A represents the time to clotting induced by E. coli LPS and B the time to clotting induced by E. coli LPS along with LPG or PG.

Statistics Data are expressed as means 5 SEM.

Results

Our experimental approach consisted in determining the effect of exogenous LPG or its entire carbohydrate fragment on coagulation time. Table 1 clearly shows that the addition of L. donovani LPG but not L. donovani PG (2pM final concentration) to human mononuclear cells incubated with E. coli LPS significantly inhibits their capacity to shorten the clotting time of normal plasma. The inhibition is dose-dependent (Figure 1). At higher concentrations of LPG (i.e. 2 pM, and 4 pM) clotting does not occur in the test tube after incubation of mononuclear cells with endotoxin (clotting time >360 seconds, 100% inhibition). Furthermore, in Figure 1 it is also shown that when the delipidated carbohydrate portion of LPG (PG) was added to the incubation mixtures, no inhibitory effect on PCA production was observed at the four concentrations tested.

Table 1 Effect of Leishmania donovani -LPG and -PG on the PCA production by mononuclear cells stimulated with Escherichia coli LPS.

Mononuclear cells No .f Clotting time % inhibition incubated with experiments (sectSEM)

E. coli LPS (0.2 Fm) 4 87.62?1.9 L. donovani LPG (2 pm) 4 >360 L. donovani PG (2 pm) 4 264.66?21.0

E. coli LPS (0.2 Fm) + L. donouani LPG (2 pm) 4 2360

E. coli LPS (0.2 wm) + L. donovani LPG (2 pm) 4 74.6626.0

100

Saline 4 >360

Discussion

Both the attachment and entry of L. donovani into the host cell are crucial for the microorganism to infect mononuclear cells. In this regard, LPG has been demonstrated to play a role in promoting the attachment of promastigotes to macrophages via C3

Del Prete, I n h i b i t i o n o f Mononuc lea r C e l l P rocoagu lan t A c t i v i t v 33

120 -

100 -

80 - c 0 .- .- 6 0 - r C -

40 -

20 -

0 , . -E 0.4 1 2 4 FM '

Figure 1 Dose-dependence of the inhibitory effect of Leishmania donovani LPG on procoagulant activity pro- duction by human mononuclear cells stimulated with Escherichia coli endotoxin.

receptors. LPG can indeed activate complement with resulting C3 deposition on the surface of the parasite (15). In our experiments, we have demon- strated that LPG possesses another activity, namely that of strongly inhibiting the in vitro production of PCA by endotoxin-stimulated human mononuclear cells. Through the extrinsic pathway of blood coagulation, PCA in turn promotes fibrin formation. This deposition of fibrin represents an important step in the aspecific defense mechanism since it contributes to the entrapment of the parasite. If entrapped within a network of fibrin, Leishmania cannot attach to monocytes and cannot be internalyzed in the phago- lysosomal system where it is protected from phagocytosis by reticulo-endothelial cells and from antimicrobial factors in the plasma if the infected circulating cells remain healthy. The mechanism of survival of Leishmania within the macrophage has been clarified by the demonstration of the LPG capacity to inhibit the activation of protein kinase C (PKC) in vitro (16). PKC is involved in the induction of the oxidative burst (17) and the phagocytosis of LPG-coated beads has been demonstrated to suppress the induction of the respiratory burst in mono- cytes (18). O n the other hand the evidence that endotoxin-induced PCA production by monocytes is dependent upon PKC activation (19) suggests that the molecular mechanism responsible for the inhibitory effect on PCA by LPG might be PKC suppression. Furthermore, in our experiments the failure of the delipidated carbohydrate portion (i.e. phosphoglycan,

PG) to inhibit PCA production is in agreement with the notion that it is the lipid domain and not the carbohydrate portion of LPG which is primarily responsible for suppression of PKC activity (20). Taken together our results suggest that LPG of L. donovani contributes to protect the parasite from fibrin entrapment.

Acknowledgments

We wish to thank Dr S. J. Turco, Department of Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky 40536, for the generous gift of LPG and PG and for his critical revision of the present paper.

This work was supported in part (40%) by a grant from the Minister0 Universiti Ricerca Scientifica Tecnologica.

Accepted 6 J u n e 1995

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