During chronic human malaria infections a profound anaemia is often present with almost undetectable levels of parasite. The concentration of reticulocytes in the
circulation is inappropriately low for the extent of the anaemia (Phillips 1986) and is not sufficient to compensate for the loss of effete and parasitised RBCs. This is accounted for by abberant cellular events in the bone marrow, the only site of erythropoiesis in humans. Under examination by light microscopy abnormalities of cell division, such as multinuclearity of normoblasts, intercytoplasmic bridging, karyorrhexis and incomplete and unequal mitotic nuclear divisions, can be seen. In man this dyserythropoietic affect of the parasite is almost certainly responsible for the majority of the anaemia, and its persistence, in chronic infections. On clearance of the parasitaemia the bone marrow
morphology returns to normal, reticulocyte counts rise and the level of haemoglobin increases (Weatherall and Abdalla, 1982; Phillips 1986).
Normal erythropoiesis starts from a pluripotent stem cell, which can develop along either the myeloid or the lymphoid line or divide to rephcate itself. During erythropoiesis the stem cell develops into a secondary stem cell (myeloid progenitor), which is then committed to the myeloid line and has hmited capacity for self renewal (figure 1.2.2.a). Following this stage are two further progenitor cells, the blast forming units (BFU-E) and then the colony forming units (CFU-B). The cell then develops through a number of precursor stages (pronormoblast, basophilic normoblast, polychromatophihc normoblast, orthochromatophilic normoblast) eventually forming a reticulocyte, which then matures into an erythrocyte in the circulation. A number of cytokines and colony stimulating factors act at various stages, making the cells proliferate and differentiate (figure
1.2.2.a). The most important of these stimulatory molecules is erythropoietin, which acts at several points of development on the erythroid line.
C eh — ^ l% g è n ito r BFU-E -^ - 4 ^ CFU-E Reticulocyte ► E rythrocyte
IL-1 IL-1 EPO EPO
11-6 IL-3 CSF CSF
IL-9 lL-9
IL-11 EPO
CSF K X y
Figure 1.2.2.a Stages of erythroid development showing the involvement of various cytokines/growth factors. CSF=Colony Stimulating Factors;
The dyserythropoietic abnormalities seen in the cellularity of the bone marrow of infected patients, are also observed in murine infections. Initially there is an overall reduction in the marrow cellularity (Maggio-Price, Brookoff & Weiss, 1985), with a decrease in pluripotent stem cells (Silverman, Schooley & Mahlmann, 1987), BFU-E and CFU-E (Maggio-Price, Brookoff & Weiss, 1985; Weiss, Johnson & Weidanz, 1989). There is some discrepancy as to whether the numbers of BFU-E are depleted or whether they just maintain the normal preinfection levels (Villeval, Lew & Metcalf,
1990). However, the overall picture in the murine bone marrow is of an initial depletion of erythroid precursors in the early stages of infection. With the development of the anaemia CFU-E cells increase, with a reduction in BFU-E levels (Yap & Stevenson, 1992: Villeval, Lew & Metcalf, 1990; Maggio-Price, Brookoff & Weiss, 1985). The subsequent precursor cells increased in number but remained below or within normal values. Therefore, the cellular events in the bone marrow indicate severe
dyerythropoiesis with only a mild increase in erythropoietic events with the development of extensive anaemia, the increase in erythropoiesis being woefully inadequate.
In humans the bone marrow is solely responsible for erythropoiesis, but in mice the marrow only serves to maintain the normal rate of RBC production. The dominant murine erythropoietic organ is the spleen (Villeval, Lew & Metcalf, 1990), which increases production in response to anaemia. Examination of splenic erythropoiesis during infection does not show the same extent of inhibition of erythropoiesis. The observed decrease in bone marrow stem cells is followed by an increase in splenic stem cells (Silverman, Schooley & Mahlmann, 1987). A corresponding increase in CFU-E and erythroblasts (normoblasts) indicated an overall increase in splenic erythropoiesis
(Villeval, Lew & Metcalf, 1990; Weiss, Johnson & Weidanz, 1989; Yap & Stevenson, 1992). This increase in splenic erythropoiesis partially compensates for the
dyserythropoiesis in the bone marrow, however, the level of RBC production by the spleen is not appropriate to the degree of anaemia. The level of erythropoiesis and resolution of the anaemia only occurs on clearance of the infection. This overview varies slightly between different murine infections, the amplification of late stages of splenic erythropoiesis is lower in fatal species of malaria (Villeval, Lew & Metcalf, 1990; Yap & Stevenson, 1992). It is suggested that this is in fact the determinant of whether the infection is lethal or not. The limited dyserythropoiesis in the spleen has been explained by the formation of a blood-spleen barrier. The locules of the filtration beds in the spleen become sealed off by activated reticular cells, this is believed to protect splenic haematopoiesis from the parasite (Weiss, Geduldig & Weidanz, 1986). It is proposed that as clearance of the parasitaemia commences, the filtration beds open and stores of reticulocytes are released to the circulation.
The lack of an appropriate degree of erythropoiesis is not due to a lowered level of erythropoietin (EPO)(figure 1.2.2.a), as is the case in certain other diseases in which anaemia occurs, such as HIV, cancer and rheumatoid arthritis (Spivak et a l, 1989; Miller et a l, 1990; Means & Krantz, 1992). The levels of EPO during malaria infection were appropriate to the extent of the anaemia (Villeval, Lew & Metcalf, 1990; Weiss & Weidanz, 1989; Silverman, Schooley & Mahlmann, 1987). Furthermore, the initial dyserythropoietic events in the marrow and the increased splenic erythropoiesis were seen to occur before the onset of a detectable anaemia (Villeval, Lew & Metcalf, 1990; Weiss & Weidanz, 1989). This indicates that the parasite does not merely inhibit the
normal erythropoietic processes that occur in response to anaemia, but directly or indirectly alters the dynamics of erythropoiesis in a pre-emptive manner.
The current evidence indicates that the dyserythropoiesis occurs indirectly due to host factors produced at a localised level in response to the presence of the parasite (Miller et a l, 1989).There is a profusion of reports implicating tumour necrosis factor (TNF) as the host derived molecule. The infusion of mice with recombinant human TNF produces the same cellular abnormalities seen during malaria (Moldawer et a l, 1989, Johnson et a l, 1989); the in vitro inhibition of human erythroid cells by TNF has also been demonstrated (Means, Dessypris & Krantz, 1990). As is the case in malaria infections, infusions of EPO were not sufficient to overcome this TNF-induced inhibition of erythropoiesis (Clibon et a l , 1990). Infected mice given either human or murine TNF develop pronounced dyserythropoiesis ( Clark & Chaudri, 1988; Miller et a l, 1989), erythropoiesis was partially restored when anti-TNF antibody was injected (Miller et a l,
1988).
Other cytokines, namely IL-1 and IFN-a, have also been demonstrated, in vitro, as capable of inhibiting erythropoietic systems (Broxmeyer et a l, 1986; Schooley,
Kullgren & Allison, 1987; Button & Levere, 1980; Mangan et a l, 1984). This has led to them being implicated in a number of infections and disorders that involve anaemia as a factor of the pathology. It should be noted that although there is a wealth of
experimental evidence supporting the involvement of a number of cytokines in the anaemia of malaria, there is a contradictory opinion that the inhibitory factor is not TNF, IFN-a or IL-1 (Yap & Stevenson, 1994). Using an in vitro system of dyserythropoiesis.
blocking antibodies to these three cytokines were not found to have any affect on the inhibitory capacity of spleen cell conditioned media from infected mice.
It is clear from the various reports that malaria parasites severely alter the erythropoietic dynamics of the host. There is still a degree of uncertainty as to the factors responsible, although there is strong evidence implicating host derived cytokines, and predominantly TNF. Dyserythropoiesis is, however, obviously a key facet of malaria-induced anaemia.