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Neurobiology of sleeping sickness
Parasitology Today, vol. 5, no. 7, I989
r
215
Neurobiology
of Sleeping Sickness
V.W. Pentreath The advanced stages ofsleeping sickness are correlated with a spread of trypanosomes into the central nervous system (CNS), producing a dissem/nated encephalitis. Inflammatory reactions extend along the blood vessels causing perivascular cuffing, which consists ofinfiltrations and proliferations oflymphocytes and also increased numbers ofastrocytes and microglia. Progress in our understanding ofthe functions of astrocytes suggests that they are efficient antigen-presenting cells, initiating and regulating the introcerebral inflammatory response and limiting parasite spread to the perivascular spaces. Sleeping sickness, or African trypanosomiasis in man, caused by 5. brucei gambiense or T. brucei rhodesiense, runs a complex course leading, if untreated, to the late-, or secondary-stage, and death. T. b. gambiense produces initially mild symptoms and a chronic infection which may last for years but T. b. rhodesiense infections are generally acute, with severe deterioration and death within a few weeks or months. The later stage is associated with the parasite becoming established within the brain, after which successful chemotherapy necessitates the use of drugs that will cross the bloodbrain barrier, otherwise t-elapses will occur. Since the 1940s such treatment has been limited to the toxic organic arsenicals (eg. Melarsoprol) which can themselves cause serious side-effects. Melarsoprol causes fatal reactive encephalopathy in up to IO% of treated patients ‘.2. Recent finding s suggest that difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, offers a hopeful alternative.
the Virchow-Robin spaces surrounding the blood vessels that enter the brain6,9. During the protracted course of the Gambian disease, the vessels become progressively surrounded by the same types of cell as the affected meninges, forming the lesion termed perivascular cuffing2,9,lo (Fig. I ). Both the cuffs and the surrounding brain parenchyma frequently contain proliferations of microglia and astrocytes. At post-mortem the vasculitis may appear obliterative, with multiple haemorrhages, especially in the midbrain and brainstem. The Immune Response Trypanosomes escape the immune response by varying their surface glycoprotein coat’ ’, thus exposing the host to altered antigenic variants, and by inducing generalized suppression of immunological function. The changes have been extensively studied in mice5. The parasite acts as a proliferative stimulus to T- and B-cells, but these become
insensitive to selection by antigen and normal control signals. The T-cell stimulation, which appears to take place only in the presence of accessory cells, does not necessarily signal the proliferation of the B-cells. The functions of all the T- and B-cell subclasses appear to be affected. Serum IgM levels are elevated throughout, although little, if any, of this antibody has activity against trypanosomes. However, despite the profound inhibition of variant-specific IgM antibodies, they are adequate to control the recurrent waves of parasitaemia up until death. Nevertheless, IgG antibody production against the parasite is suppressed much earlier in the infection. It is not known how the parasite renders the T- or B-cells refractory to selection by antigen, leaving the B-cells to mature into production of large amounts of nonspecific Ig molecules. It is unlikely to be clonal exhaustion because of the speed at whrch the suppression takes place in acute infections. Also, trypanosomes, or their products, do not appear to act directly on T- or B-cells. Alternatively it has been shown that macrorespond markedly during phages infection by releasing interleukin- I (IL- I ) and prostaglandin E2 (PGEl)‘. Other cells release intetferons. Thus the reasons may lie in changes induced in other cell types, causing, for example, alterations in lymphokines which in turn
Neuropathology The mouse model has been especially valuable for studying the CNS involvement in sleeping sickness3-5. The parasite may enter the CNS via the choroid4,6, via regions in which the blood-brain barrier is incomplete7, or through transient leaks in the barrier’. It may then be carried in the cerebrospinal fluid (CSF) circulating through the subarachnoidal spaces over the surface of the brain, from where it can move inwards along the perivascular extensions (Virchow-Robin spaces)6. The inflammation affects the pia-arachnoid, which becomes thickened due to the infiltration of lymphocytes (especially Bcells), plasma cells and so-called morular cells’. Morular cells are late stages of the immature lymphocyte-mature plasma cell series containing large aggregates of immunoglobulin, which they secrete. The inflammation subsequently invades
Elsewr Sc,enre Publtihers 0 ,989.
Ltd. (UK) 0 l694707/891803 50
of
fig. 1. A blood vessel in the thalamus a fota/ case of sleeping sickness. The vessel is heavily cuffed with perivascular infiltration of lymphocytes and plasma cells. The cells may be thirty deep in advanced cases (haematoxylin and eosin stain). Reprinted from Ref 2.
Parasitology Today, vol. 5, no. 7, I989
216
Aef
fig. 2. Astrocytes and the inflammatory response in the CNS. The micrograph shows ostrocytes in human cortex (Cojol silver stain). Note the thin ostrocyte processes (some orrowed) extending to the blood vessels. The diagram beneath the micrograph shows the relation between capillary (C), ostrocytes (As) and neuron (NJ. The arrows mark end-feet The diagram on the right shows the blood-brain interface in central nervous tissue. The endotheliol cells (En) form o monolayer with adjacent cell membranes joined by o belt oftight junctions, which is the physical basis ofthe blood-brain barrier. fericytes (P) ore scattered along, and commonly enclosed by, the basement membrane (Sm). Pericytes may be microgliol cell precursors, or they might hove o role in the immune response, perhaps o&g OSadditional antigen-presenting cells. The ostrocyte end-feet (Aefl ore joined by gap junctions to form D continuous layer woven around the blood vessel. Activated T-cells (7) ore able to cross the endothelium and basement membrane. This takes place via direct migration through the endofhelium (emperipolesis) or perhaps through altered tight junctions. Once the lymphocytes reach the CNS side ofthe basement membrane they immediately make contact with the ostrocyte end-feet lfporosites enter the parenchymo, astrocytes would be well positioned to transport (P) and present antigens. The subsequent secretion ofmedioton (including IL- 1 ond L-3, interferon and PGE) by ostrocytes and lymphoctyes may (I) sustain the local inflammatory response with lymphocyte proliferation or suppression, (2) promote loco/ ostrocyte proliferation, pericyte development into microglio or a@~ other structures locally in the porenchymo to produce disease symptoms (eg. somnolence), or (3) enter the blood to promote further lymphocyte entry via a reduced or damaged blood-broin barrier.
upset the behaviour and interactions of the T- and B-ceils. The immune changes within the CNS are little understood, largely because the bases for the normal immune status of this tissue have only recently been unravelled. In health, IgM is almost absent from the CSF, but in the late stage of
sleeping sickness it becomes markedly elevated. It is produced inside the brain (by the morular cells, among others) rather than entering via damage to the blood-brain barrier. Immune complexes are formed between the variant antigens of the parasite, and antibody. These are found in the CSF” and if
deposited in the brain could cause damage to small vessels, and localized anaemia. However, the general absence of infiltration with polymorphonuclear neutrophils argues against this, as does much of the clinical picture of Gambian sleeping sickness’3. It is also very unlikely that the brain damage is a delayed
hrasitology
Today, vol. 5, no. 7, I989
hypersensitivity type reaction since the infiltrating lymphocytes are chiefly of the B-type. Searches have been made for immunopathological alterations in the CNS. Sera of some infected mice may contain antibodies against Ineurons ofthe hippocampus and hypothalamusi4, but other studies have failed to detect any antl-neuronal antibodies. However, apart from some local chromatolysis of neurons around blood vessels in some chronic cases’, there is a surprising absence of neuronal damage in sleeping sickness. A more feasible Interpretation is that there is an immunoproliferative disorder with massive B-oell invasion of the perrvascular spaces and Ig synthesis within the CNS13.This seems more likely if considered in relation to recent information outlined below on the functions of astrocytes. Astrocytes and Late-stage Sleeping Sickness The traditional view that -the CNS is an immunologically privileged site, without lymphatic drainage and relqdered inaccessible to the immune system by the blood-brain barrier, is currently being drastically re-evaluated. It is now clear that activated (but not resting) T-cells can cross the endothelial blood-brain barrier and that some glial cells, primarily the astrocytes, are vital partner-s in a complex control interface between the immune and the nervous systems. The properties of the control interface seem beautifully designed to protect central nervous tissue. Because the delicate arrangement of neurons and synapses in the CNS parenchyma is more vulnerable and has very low regenerat:ive capacity compared with other tissues, it is essential not only that any infective organism should be quickly eliminated, but also that any immune reactions should be kept to a minimum. This appears to be achieved by restricting the number of lymphocytes entering the CNS and by focusing their responses on the perivascular areas. The interface between the blood and the central nervous tissue consists of concentric layers of endothelial cells, basal membranes and astrocyte endfeet (Fig. 2). Astrocytes are one of the most numerous cell types in the CNS. Their structure is unusual in that their nuclear regions may be localized at a distance from the blood vessels, but they give rise to radiating processes which may extend across vast distances of parenchyma to make contact with blood
217
‘He seems to hove solved the problem blood-brain
of the
barrier.’
vessels, or with different neurons or synapses (Fig. 2). These cells are involved in a variety of supportive, homeostatic, phagocytic and trophic functions which are essential for the well-being of the neurons (eg. Ref. IS). They also cooperate with antigen-specific T-cells to control immune reactivity within the CNS16. This information has been obtained from a range of in vitro experiments with cultured astrocytes as well as studies of inflammatory CNS diseases using murine models, and appears to be directly relevant to sleeping sickness. In contrast to most body tissues, both class I and class II antigens of the major histocompatibility complex (MHC) are absent or only minimally expressed in healthy CNS parenchyma. However, this situation can be readily reversed by pathogens and pathological conditions. Activated T-cells cross the blood-brain barrier and interact with astrocytes by releasing factors, including y-interferon (IFN-y), which induce the astrocytes to synthesize and express on their membranes class II MHC antigens (la determinants)16. la-induced astrocytes are efficient antigen-presenting cells (APCs). Soluble products released from actvated T-cells stimulate astrocyte proliferation. Brain endothelial cells can also express class II MHC I’. Astrocytes can be induced to manufacture and secrete IL- I and IL-3, interferon, and PGE’*,‘9. Theamounts of IL- I and PGE produced in experimental culture systems may be exceptionally large. Moreover, astrocytes can strongly stimulate antigen-reactive T-cells, and in some situations are capable of suppressing T-cell activation. The suppression can be modulated by IFN-Y’~. Thus astrocytes are emerging as the primary facultative APCs regulating immune reactivity in CNS parenchyma. Their ubiquitous distribution and special
arrangement at the blood-brain interface would appear to be designed perfectly for collecting and then concentrating antigenic determinants around blood vessels, therefore protecting the parenchyma by focusing the immune reactions and allowing rapid clearing of toxic metabolites in the blood. Because la expression must be actively induced, the reactions will cease when the local production of stimulating factors is stopped I6 The capacity of astrocytes to produce immunosuppressive mediators such as PGE appears additionally impot-tant in potentially limiting the immune response. Much research will be necessary in order to understand late-stage sleeping sickness in the light of the new insights into the immune regulation of the CNS. However, it may be hypothesized that activated T-cells will cross the bloodbrain barrier and induce astrocytes to synthesize la determinants throughout the CNS at a very early stage of the disease and this will be maintained throughout its course. The parasite is inhibited from penetrating the parenthyme because of the blood-brain barrier and unfavourable composition of the CSF, but progressively enters via various opportunistic routes (eg. capillary leaks or Virchow-Robin spaces). The affected astrocytes then interact locally with the activated T-cells to produce cytokines, which in turn may sustain proliferation of lymphocytes, astrocytes or microglia, and induce a concomitant B-cell response and antibody secretion (Fig. 2). Alternatively, depending on, for example, the degree of receptor activation in the astrocytes, there may be secretion of prostaglandins, and immunosuppression. Cellular infiltration and further parasite entry associated wrth blood-brain barrier breakdown will take place at sites of inflammation and this will be perpetuated by successive antigenic changes of the parasite. The course of the disease and its cerebral manifestations WIII depend on the sites of inflammation, the interactions between parasites, antibodies and astrocytes and the levels and types of mediator release at these sites. The situation is comparable to the macrophagemediated alterations that have been suggested to contribute to immune dysfunctions elsewhere in the body’. It should be noted that IL- I has a variety of effects in brain, including potent fever and slowwave sleep-inducing activities2’. Active production of IL-I and other factors inside the blood-brain barrier probably produce the symptoms giving sleeping sickness its name.
Parasitology Today, vol. 5, no.7, I989
218
The ability of the parasite to evade and suppress the immune system, together with the complex barrier systems and immune responses of central nervous tissue, make the development of suitable agents for the chemotherapy of late-stage sleeping sickness an almost Herculean task. A proper understanding of the underlying cellular and immune changes in the CNS are, however, essential for progress in this direction. References I De Raadt, P. ( I984)Med. Int. 2, I46- I50 2 Adams, J.H. et al. (I 986) Neuropothol. Neurobrol. I 2,8 i-94
Appl.
3 Jennings. F.W. et al. (1979) fnt. J, Porasrtol. 9, 38 I-384 4 Polterra, A.A. et al. ( 1980) Clm. Exp. Immunol. 40,496-507 5 Askonas, B.A. and Bancroft, G.]. ( 1984) Phrlos. Trans. R. Sot. London Ser. B 307,4 l-50 6 Schmidt, H. (I 983) VirchoM Arch. Abt. A Pathol. Anat. 399,333-343 7 Schultzberg, M. et al. ( I988)/. Neurosci. Res. 2 I, s&6 8 Nagy. Z., Peters, H. and Huttner, I. (I 983) lob. Invest. 49,662-67 I 9 Mott, F.W. ( I 906) Rep. Sleeping SrcknessComm. R. Sot. 7,3-46 IO Ormerod, W.E. (I 970) in The Afrrcan Jrypanosomiases (Mulligan, H.W., ed.). pp 587-60 I, Allen and Unwin I I Cross, G.A.M. (I 984) Philos. Trans. R. Sot. London Ser. 6 307,3- I2
I2 Lambert P.H.. Berney, M. and Kazyumba. G. (I 98 I )I. Clm. Invest. 67,77-85 I3 Greenwood, B.M. and Whittle, H.C. (1980) Trans. R. Sot. Jrop. Med. Hyg. 74,7 16-725 I4 Polterra. A.A. (I 980) Trans. R. Sot. Jr@. Med. Hyg. 74,706-7 I5 I5 Pentreath. V.W. (I 982) Trends Neuroscr. 5, 339-345 I6 Wekerle, H. et al. (I 987)). i!%p.6101.I32,43-57 I7 McCarron, R.M. et al. (I 985)j. Immunol. 134, lO(&lO3 18 Fontana. A. et al. C1982) 1. Immunol. 129. 2413-2419 ’ ” I9 Frei. K. et al. (I 985)j. Immunol. I35,4044-4047 20 Kreuger, J.M. et al: (I 984) Am. J. Phyziof. 246, R994R999 Victor Pentreath
of BioSalford, Salford
is at the Department
logical Sciences,University
of
M5 4WT, UK.
Red Cell Deformability and Invasion by Malaria Parasites G. Pasvol and R.J.M. Wilson Malaria parasites enter red cells in a rnultimembrane deformation, invagination and encapsulation. The molecular basis of red cell rigidity is examined by GeoffPasvol and lain Wilson, and they discuss its efect on the efficiency of invasion by various Plasmodium spp.
step process involving attachment,
Invasion of red cells by malaria parasites does not involve penetration of the host cell membrane. Instead, a multi-step process induces the membrane to deform during at least three stages of red cell entry ‘.2. Upon attachment of the invading merozoite to the cell there is a transient but widespread perturbation of the membrane which is followed within a few seconds by a localized invagination in which the parasite is ultimately encapsulated. Following interiorization in P. knowlesi infections, a final wave of deformation involves the entire cell. The internalized membrane surrounding the parasite may contain both host and parasite components but the number of intramembrane particles (IMPS) is, at least initially, greatly reduced3. Because the usual cytoskeletal components ofthe host cell are absent from the parasitophorous vacuolar membrane4, it must be regarded, at best, as a grossly modified form ofthe host cell membrane. While these lines of evidence provide a visual description of membrane deformation during parasite entry, they fail to answer the question as to whether deformation events are necessary for invasion to occur. Invasion of human cells by P. folciparum, for example, does not appear to be followed by deformation of the whole cell after interiorization has
occurred as in the case of P. knowlesi. This would indicate that this phase of deformation may not be crucial in the invasion process5. Thus the relationship of deformation to parasite invasion remains unclear. Deformability and its Measurement By defining more precisely the molecular changes underlying invasion-
inhibition systems, and by measuring the effect of bound ligands or chemical perturbations on ‘deformability’, an assessment can be made in quantitative terms of red cell properties that affect deformability and invasion. However, in order to draw any useful conclusions about the relation of the efficiency of malarial invasion to membrane deformability, a clear definition is required. Various methods have been devised for observing the mechanical properties of the red cell membrane and
Box I. Glossaryof Terms Celldeformability. This is the capacity of the cell to change its shape under applied stress. Ektacytometry and Couette viscometry can give an indication of whole cell deformability which in turn depends on the viscoelastic properties of the membrane, the viscosity of the cytosol, and on cell shape. Shear viscosity.The red cell membrane exhibits both elastic and plastic deformation and each of these has a characteristic shear viscosity. Viscoelastic behaviour is defined by the dissipation of internal viscous energy (nominally 0. I pN s-’ cm-‘), whereas plastic behaviour is reflected in irrecoverable extension after the elastic limit of the membrane has been exceeded (nominally I pN cm-‘). Shear elastic modulus. This can be defined as the recoverable hyperelastic response of the membrane and is usually measured by aspiration (nominally I pN cm-‘). Discocyte. A normal red cell has a biconcave discoid shape as its minimum energy contour, since micromechanical measurements indicate no appreciable stress in the membrane in this resting state. During circulation, normal red cells constantlychange shape in response to a range of dynamic shear stresses. Hereditary spherocytosis. This is the most common inherited haemolytic anaemia in Caucasians, characterized by red cells with stomatocytic or spherical shape and increased osmotic fragility due to instability of the membrane. Hereditary pyropoikilocytosis.This is a rare disease which presents in early childhood as a severe haemolytic anaemia characterized by thermal instability of the membrane resulting in budding red cells and marked fragmentation. @ 1989, Elsewr
Saence Publnhers Ltd. (UK) 0 I694707/891803
50
r
215
Neurobiology
of Sleeping Sickness
V.W. Pentreath The advanced stages ofsleeping sickness are correlated with a spread of trypanosomes into the central nervous system (CNS), producing a dissem/nated encephalitis. Inflammatory reactions extend along the blood vessels causing perivascular cuffing, which consists ofinfiltrations and proliferations oflymphocytes and also increased numbers ofastrocytes and microglia. Progress in our understanding ofthe functions of astrocytes suggests that they are efficient antigen-presenting cells, initiating and regulating the introcerebral inflammatory response and limiting parasite spread to the perivascular spaces. Sleeping sickness, or African trypanosomiasis in man, caused by 5. brucei gambiense or T. brucei rhodesiense, runs a complex course leading, if untreated, to the late-, or secondary-stage, and death. T. b. gambiense produces initially mild symptoms and a chronic infection which may last for years but T. b. rhodesiense infections are generally acute, with severe deterioration and death within a few weeks or months. The later stage is associated with the parasite becoming established within the brain, after which successful chemotherapy necessitates the use of drugs that will cross the bloodbrain barrier, otherwise t-elapses will occur. Since the 1940s such treatment has been limited to the toxic organic arsenicals (eg. Melarsoprol) which can themselves cause serious side-effects. Melarsoprol causes fatal reactive encephalopathy in up to IO% of treated patients ‘.2. Recent finding s suggest that difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, offers a hopeful alternative.
the Virchow-Robin spaces surrounding the blood vessels that enter the brain6,9. During the protracted course of the Gambian disease, the vessels become progressively surrounded by the same types of cell as the affected meninges, forming the lesion termed perivascular cuffing2,9,lo (Fig. I ). Both the cuffs and the surrounding brain parenchyma frequently contain proliferations of microglia and astrocytes. At post-mortem the vasculitis may appear obliterative, with multiple haemorrhages, especially in the midbrain and brainstem. The Immune Response Trypanosomes escape the immune response by varying their surface glycoprotein coat’ ’, thus exposing the host to altered antigenic variants, and by inducing generalized suppression of immunological function. The changes have been extensively studied in mice5. The parasite acts as a proliferative stimulus to T- and B-cells, but these become
insensitive to selection by antigen and normal control signals. The T-cell stimulation, which appears to take place only in the presence of accessory cells, does not necessarily signal the proliferation of the B-cells. The functions of all the T- and B-cell subclasses appear to be affected. Serum IgM levels are elevated throughout, although little, if any, of this antibody has activity against trypanosomes. However, despite the profound inhibition of variant-specific IgM antibodies, they are adequate to control the recurrent waves of parasitaemia up until death. Nevertheless, IgG antibody production against the parasite is suppressed much earlier in the infection. It is not known how the parasite renders the T- or B-cells refractory to selection by antigen, leaving the B-cells to mature into production of large amounts of nonspecific Ig molecules. It is unlikely to be clonal exhaustion because of the speed at whrch the suppression takes place in acute infections. Also, trypanosomes, or their products, do not appear to act directly on T- or B-cells. Alternatively it has been shown that macrorespond markedly during phages infection by releasing interleukin- I (IL- I ) and prostaglandin E2 (PGEl)‘. Other cells release intetferons. Thus the reasons may lie in changes induced in other cell types, causing, for example, alterations in lymphokines which in turn
Neuropathology The mouse model has been especially valuable for studying the CNS involvement in sleeping sickness3-5. The parasite may enter the CNS via the choroid4,6, via regions in which the blood-brain barrier is incomplete7, or through transient leaks in the barrier’. It may then be carried in the cerebrospinal fluid (CSF) circulating through the subarachnoidal spaces over the surface of the brain, from where it can move inwards along the perivascular extensions (Virchow-Robin spaces)6. The inflammation affects the pia-arachnoid, which becomes thickened due to the infiltration of lymphocytes (especially Bcells), plasma cells and so-called morular cells’. Morular cells are late stages of the immature lymphocyte-mature plasma cell series containing large aggregates of immunoglobulin, which they secrete. The inflammation subsequently invades
Elsewr Sc,enre Publtihers 0 ,989.
Ltd. (UK) 0 l694707/891803 50
of
fig. 1. A blood vessel in the thalamus a fota/ case of sleeping sickness. The vessel is heavily cuffed with perivascular infiltration of lymphocytes and plasma cells. The cells may be thirty deep in advanced cases (haematoxylin and eosin stain). Reprinted from Ref 2.
Parasitology Today, vol. 5, no. 7, I989
216
Aef
fig. 2. Astrocytes and the inflammatory response in the CNS. The micrograph shows ostrocytes in human cortex (Cojol silver stain). Note the thin ostrocyte processes (some orrowed) extending to the blood vessels. The diagram beneath the micrograph shows the relation between capillary (C), ostrocytes (As) and neuron (NJ. The arrows mark end-feet The diagram on the right shows the blood-brain interface in central nervous tissue. The endotheliol cells (En) form o monolayer with adjacent cell membranes joined by o belt oftight junctions, which is the physical basis ofthe blood-brain barrier. fericytes (P) ore scattered along, and commonly enclosed by, the basement membrane (Sm). Pericytes may be microgliol cell precursors, or they might hove o role in the immune response, perhaps o&g OSadditional antigen-presenting cells. The ostrocyte end-feet (Aefl ore joined by gap junctions to form D continuous layer woven around the blood vessel. Activated T-cells (7) ore able to cross the endothelium and basement membrane. This takes place via direct migration through the endofhelium (emperipolesis) or perhaps through altered tight junctions. Once the lymphocytes reach the CNS side ofthe basement membrane they immediately make contact with the ostrocyte end-feet lfporosites enter the parenchymo, astrocytes would be well positioned to transport (P) and present antigens. The subsequent secretion ofmedioton (including IL- 1 ond L-3, interferon and PGE) by ostrocytes and lymphoctyes may (I) sustain the local inflammatory response with lymphocyte proliferation or suppression, (2) promote loco/ ostrocyte proliferation, pericyte development into microglio or a@~ other structures locally in the porenchymo to produce disease symptoms (eg. somnolence), or (3) enter the blood to promote further lymphocyte entry via a reduced or damaged blood-broin barrier.
upset the behaviour and interactions of the T- and B-ceils. The immune changes within the CNS are little understood, largely because the bases for the normal immune status of this tissue have only recently been unravelled. In health, IgM is almost absent from the CSF, but in the late stage of
sleeping sickness it becomes markedly elevated. It is produced inside the brain (by the morular cells, among others) rather than entering via damage to the blood-brain barrier. Immune complexes are formed between the variant antigens of the parasite, and antibody. These are found in the CSF” and if
deposited in the brain could cause damage to small vessels, and localized anaemia. However, the general absence of infiltration with polymorphonuclear neutrophils argues against this, as does much of the clinical picture of Gambian sleeping sickness’3. It is also very unlikely that the brain damage is a delayed
hrasitology
Today, vol. 5, no. 7, I989
hypersensitivity type reaction since the infiltrating lymphocytes are chiefly of the B-type. Searches have been made for immunopathological alterations in the CNS. Sera of some infected mice may contain antibodies against Ineurons ofthe hippocampus and hypothalamusi4, but other studies have failed to detect any antl-neuronal antibodies. However, apart from some local chromatolysis of neurons around blood vessels in some chronic cases’, there is a surprising absence of neuronal damage in sleeping sickness. A more feasible Interpretation is that there is an immunoproliferative disorder with massive B-oell invasion of the perrvascular spaces and Ig synthesis within the CNS13.This seems more likely if considered in relation to recent information outlined below on the functions of astrocytes. Astrocytes and Late-stage Sleeping Sickness The traditional view that -the CNS is an immunologically privileged site, without lymphatic drainage and relqdered inaccessible to the immune system by the blood-brain barrier, is currently being drastically re-evaluated. It is now clear that activated (but not resting) T-cells can cross the endothelial blood-brain barrier and that some glial cells, primarily the astrocytes, are vital partner-s in a complex control interface between the immune and the nervous systems. The properties of the control interface seem beautifully designed to protect central nervous tissue. Because the delicate arrangement of neurons and synapses in the CNS parenchyma is more vulnerable and has very low regenerat:ive capacity compared with other tissues, it is essential not only that any infective organism should be quickly eliminated, but also that any immune reactions should be kept to a minimum. This appears to be achieved by restricting the number of lymphocytes entering the CNS and by focusing their responses on the perivascular areas. The interface between the blood and the central nervous tissue consists of concentric layers of endothelial cells, basal membranes and astrocyte endfeet (Fig. 2). Astrocytes are one of the most numerous cell types in the CNS. Their structure is unusual in that their nuclear regions may be localized at a distance from the blood vessels, but they give rise to radiating processes which may extend across vast distances of parenchyma to make contact with blood
217
‘He seems to hove solved the problem blood-brain
of the
barrier.’
vessels, or with different neurons or synapses (Fig. 2). These cells are involved in a variety of supportive, homeostatic, phagocytic and trophic functions which are essential for the well-being of the neurons (eg. Ref. IS). They also cooperate with antigen-specific T-cells to control immune reactivity within the CNS16. This information has been obtained from a range of in vitro experiments with cultured astrocytes as well as studies of inflammatory CNS diseases using murine models, and appears to be directly relevant to sleeping sickness. In contrast to most body tissues, both class I and class II antigens of the major histocompatibility complex (MHC) are absent or only minimally expressed in healthy CNS parenchyma. However, this situation can be readily reversed by pathogens and pathological conditions. Activated T-cells cross the blood-brain barrier and interact with astrocytes by releasing factors, including y-interferon (IFN-y), which induce the astrocytes to synthesize and express on their membranes class II MHC antigens (la determinants)16. la-induced astrocytes are efficient antigen-presenting cells (APCs). Soluble products released from actvated T-cells stimulate astrocyte proliferation. Brain endothelial cells can also express class II MHC I’. Astrocytes can be induced to manufacture and secrete IL- I and IL-3, interferon, and PGE’*,‘9. Theamounts of IL- I and PGE produced in experimental culture systems may be exceptionally large. Moreover, astrocytes can strongly stimulate antigen-reactive T-cells, and in some situations are capable of suppressing T-cell activation. The suppression can be modulated by IFN-Y’~. Thus astrocytes are emerging as the primary facultative APCs regulating immune reactivity in CNS parenchyma. Their ubiquitous distribution and special
arrangement at the blood-brain interface would appear to be designed perfectly for collecting and then concentrating antigenic determinants around blood vessels, therefore protecting the parenchyma by focusing the immune reactions and allowing rapid clearing of toxic metabolites in the blood. Because la expression must be actively induced, the reactions will cease when the local production of stimulating factors is stopped I6 The capacity of astrocytes to produce immunosuppressive mediators such as PGE appears additionally impot-tant in potentially limiting the immune response. Much research will be necessary in order to understand late-stage sleeping sickness in the light of the new insights into the immune regulation of the CNS. However, it may be hypothesized that activated T-cells will cross the bloodbrain barrier and induce astrocytes to synthesize la determinants throughout the CNS at a very early stage of the disease and this will be maintained throughout its course. The parasite is inhibited from penetrating the parenthyme because of the blood-brain barrier and unfavourable composition of the CSF, but progressively enters via various opportunistic routes (eg. capillary leaks or Virchow-Robin spaces). The affected astrocytes then interact locally with the activated T-cells to produce cytokines, which in turn may sustain proliferation of lymphocytes, astrocytes or microglia, and induce a concomitant B-cell response and antibody secretion (Fig. 2). Alternatively, depending on, for example, the degree of receptor activation in the astrocytes, there may be secretion of prostaglandins, and immunosuppression. Cellular infiltration and further parasite entry associated wrth blood-brain barrier breakdown will take place at sites of inflammation and this will be perpetuated by successive antigenic changes of the parasite. The course of the disease and its cerebral manifestations WIII depend on the sites of inflammation, the interactions between parasites, antibodies and astrocytes and the levels and types of mediator release at these sites. The situation is comparable to the macrophagemediated alterations that have been suggested to contribute to immune dysfunctions elsewhere in the body’. It should be noted that IL- I has a variety of effects in brain, including potent fever and slowwave sleep-inducing activities2’. Active production of IL-I and other factors inside the blood-brain barrier probably produce the symptoms giving sleeping sickness its name.
Parasitology Today, vol. 5, no.7, I989
218
The ability of the parasite to evade and suppress the immune system, together with the complex barrier systems and immune responses of central nervous tissue, make the development of suitable agents for the chemotherapy of late-stage sleeping sickness an almost Herculean task. A proper understanding of the underlying cellular and immune changes in the CNS are, however, essential for progress in this direction. References I De Raadt, P. ( I984)Med. Int. 2, I46- I50 2 Adams, J.H. et al. (I 986) Neuropothol. Neurobrol. I 2,8 i-94
Appl.
3 Jennings. F.W. et al. (1979) fnt. J, Porasrtol. 9, 38 I-384 4 Polterra, A.A. et al. ( 1980) Clm. Exp. Immunol. 40,496-507 5 Askonas, B.A. and Bancroft, G.]. ( 1984) Phrlos. Trans. R. Sot. London Ser. B 307,4 l-50 6 Schmidt, H. (I 983) VirchoM Arch. Abt. A Pathol. Anat. 399,333-343 7 Schultzberg, M. et al. ( I988)/. Neurosci. Res. 2 I, s&6 8 Nagy. Z., Peters, H. and Huttner, I. (I 983) lob. Invest. 49,662-67 I 9 Mott, F.W. ( I 906) Rep. Sleeping SrcknessComm. R. Sot. 7,3-46 IO Ormerod, W.E. (I 970) in The Afrrcan Jrypanosomiases (Mulligan, H.W., ed.). pp 587-60 I, Allen and Unwin I I Cross, G.A.M. (I 984) Philos. Trans. R. Sot. London Ser. 6 307,3- I2
I2 Lambert P.H.. Berney, M. and Kazyumba. G. (I 98 I )I. Clm. Invest. 67,77-85 I3 Greenwood, B.M. and Whittle, H.C. (1980) Trans. R. Sot. Jrop. Med. Hyg. 74,7 16-725 I4 Polterra. A.A. (I 980) Trans. R. Sot. Jr@. Med. Hyg. 74,706-7 I5 I5 Pentreath. V.W. (I 982) Trends Neuroscr. 5, 339-345 I6 Wekerle, H. et al. (I 987)). i!%p.6101.I32,43-57 I7 McCarron, R.M. et al. (I 985)j. Immunol. 134, lO(&lO3 18 Fontana. A. et al. C1982) 1. Immunol. 129. 2413-2419 ’ ” I9 Frei. K. et al. (I 985)j. Immunol. I35,4044-4047 20 Kreuger, J.M. et al: (I 984) Am. J. Phyziof. 246, R994R999 Victor Pentreath
of BioSalford, Salford
is at the Department
logical Sciences,University
of
M5 4WT, UK.
Red Cell Deformability and Invasion by Malaria Parasites G. Pasvol and R.J.M. Wilson Malaria parasites enter red cells in a rnultimembrane deformation, invagination and encapsulation. The molecular basis of red cell rigidity is examined by GeoffPasvol and lain Wilson, and they discuss its efect on the efficiency of invasion by various Plasmodium spp.
step process involving attachment,
Invasion of red cells by malaria parasites does not involve penetration of the host cell membrane. Instead, a multi-step process induces the membrane to deform during at least three stages of red cell entry ‘.2. Upon attachment of the invading merozoite to the cell there is a transient but widespread perturbation of the membrane which is followed within a few seconds by a localized invagination in which the parasite is ultimately encapsulated. Following interiorization in P. knowlesi infections, a final wave of deformation involves the entire cell. The internalized membrane surrounding the parasite may contain both host and parasite components but the number of intramembrane particles (IMPS) is, at least initially, greatly reduced3. Because the usual cytoskeletal components ofthe host cell are absent from the parasitophorous vacuolar membrane4, it must be regarded, at best, as a grossly modified form ofthe host cell membrane. While these lines of evidence provide a visual description of membrane deformation during parasite entry, they fail to answer the question as to whether deformation events are necessary for invasion to occur. Invasion of human cells by P. folciparum, for example, does not appear to be followed by deformation of the whole cell after interiorization has
occurred as in the case of P. knowlesi. This would indicate that this phase of deformation may not be crucial in the invasion process5. Thus the relationship of deformation to parasite invasion remains unclear. Deformability and its Measurement By defining more precisely the molecular changes underlying invasion-
inhibition systems, and by measuring the effect of bound ligands or chemical perturbations on ‘deformability’, an assessment can be made in quantitative terms of red cell properties that affect deformability and invasion. However, in order to draw any useful conclusions about the relation of the efficiency of malarial invasion to membrane deformability, a clear definition is required. Various methods have been devised for observing the mechanical properties of the red cell membrane and
Box I. Glossaryof Terms Celldeformability. This is the capacity of the cell to change its shape under applied stress. Ektacytometry and Couette viscometry can give an indication of whole cell deformability which in turn depends on the viscoelastic properties of the membrane, the viscosity of the cytosol, and on cell shape. Shear viscosity.The red cell membrane exhibits both elastic and plastic deformation and each of these has a characteristic shear viscosity. Viscoelastic behaviour is defined by the dissipation of internal viscous energy (nominally 0. I pN s-’ cm-‘), whereas plastic behaviour is reflected in irrecoverable extension after the elastic limit of the membrane has been exceeded (nominally I pN cm-‘). Shear elastic modulus. This can be defined as the recoverable hyperelastic response of the membrane and is usually measured by aspiration (nominally I pN cm-‘). Discocyte. A normal red cell has a biconcave discoid shape as its minimum energy contour, since micromechanical measurements indicate no appreciable stress in the membrane in this resting state. During circulation, normal red cells constantlychange shape in response to a range of dynamic shear stresses. Hereditary spherocytosis. This is the most common inherited haemolytic anaemia in Caucasians, characterized by red cells with stomatocytic or spherical shape and increased osmotic fragility due to instability of the membrane. Hereditary pyropoikilocytosis.This is a rare disease which presents in early childhood as a severe haemolytic anaemia characterized by thermal instability of the membrane resulting in budding red cells and marked fragmentation. @ 1989, Elsewr
Saence Publnhers Ltd. (UK) 0 I694707/891803
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