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Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of B cells in leishmaniasis has been addressed in models of cutaneous leishmaniasis. Continual administration of anti-IgM Abs, which causes B cell depletion, enhanced resistance to Leishmania tropica and Leishmania mexicana in BALB/c mice (7). BALB.xid mice, which lack B-1 B cells and have a marked reduction in B-2 B cell number, also exhibit enhanced resistance to Leishmania major infection (8). More recently, it has been shown that cotransfer of B cells converts T cell-reconstituted L. major-resistant, C.B-17 scid mice into a susceptible phenotype (9). Similarly, administration of IL-7, a B cell hematopoietic factor, was shown to markedly increase B cell number and exacerbate L. major infection (10). In contrast to these data, in a study using gene-targeted mice, no evidence was found for a contribution of B lymphocytes to the development of polarized Th responses to L. major in either genetically resistant or susceptible mice (11).
In the present study, B cell-deficient IgM transmembrane domain (µMT) mutant mice were used to investigate the role of B lymphocytes in murine VL caused by infection with L. donovani. Our data indicate that 1) in the genetic absence of B cells, mice show enhanced resistance to L. donovani, but with associated destructive hepatic pathology; 2) neutrophils play a key role in host resistance to L. donovani in both B cell-deficient and wild-type mice; and 3) in B cell-deficient mice, serum transfer protects against exaggerated pathology without altering the heightened level of resistance seen in these mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female µMT and C57BL/6 mice were used at 6 to 8 wk of age. C57BL/6 (Nramp1s (12, 13)) mice were purchased from Bantin & Kingman (Hull, U.K.). C57BL/6 recombinase activating gene 1 (RAG1-/-) mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME), and 4µMT mice (14) were originally obtained from Bantin & Kingman. These strains were bred at the London School of Hygiene and Tropical Medicine under barrier conditions. µMT mice were backcrossed six generations on the C57BL/6 background. The Ethiopian LV9 strain of L. donovani was used in these experiments and was maintained by passage in Syrian hamsters and purified as described elsewhere (5). Mice were infected i.v. via the lateral tail vein with 2 x 107 amastigotes in 200 µl of RPMI 1640. The course of visceral infection was determined by examining methanol-fixed, Giemsa-stained imprints of the cut spleen and liver, and quantitating organ parasite burdens as Leishman-Donovan units (LDU) using the formula: LDU = [(number of parasites/1000 host nuclei) x organ weight in milligrams (mg)]. Induction of hepato-splenomegaly was assessed using liver and spleen indices, calculated using the formula: Organ index = (organ weight in mg/total body weight in mg) x 100.
Adoptive transfer of T cells
Mice were killed by cervical dislocation and spleens mechanically disrupted by passing through a nylon sieve. Erythrocytes were lysed by treatment with Tris-buffered ammonium chloride (0.747% (w/v) NH4Cl and 0.017 M Tris, pH 7.5). Cells were then washed with MACS buffer (PBS, pH 7.2, with 0.01% (w/v) NaN3, 1% (w/v) BSA, and 5 mM EDTA), and incubated with a mixture of anti-CD4 and anti-CD8 magnetic beads (5 µl each/107 cells; Miltenyi Biotec , Surrey, U.K.) for 15 min at 12°C. After washing and resuspension at 108 cells/ml, cells were positively selected on a MACS column (Miltenyi Biotec). Purity of cells was assessed by FACS using anti-B220-FITC (clone RA3-6B2; PharMingen, San Diego, CA), anti-CD4-PE (clone H129.19) and anti-CD8-FITC (clone 53-6.7) (Sigma, Poole, U.K.). T lymphocyte populations selected for both CD4 and CD8 expression contained <1% CD4- and CD8- cells. Unfractionated spleen cells were also analyzed by flow cytometry, as above. A total of 1 x 105 or 1 x 104 of the mixed population of CD4+ and CD8+ T cells, derived from either naive µMT or C57BL/6 mice, were transferred to C57BL/6 RAG1-/- mice in 200 µl RPMI 1640. All mice were infected with L. donovani 1 day after transfer.
Adoptive transfer of serum
Serum was obtained by cardiac puncture from naive C57BL/6 and from C57BL/6 mice infected for 56 days with L. donovani. Before transfer, serum was centrifuged at 100,000 x g for 30 min at 4°C in a Beckman (Fullerton, CA) TL-100 ultracentrifuge. Two hundred microliters of normal serum or chronic-infection serum was given i.p. to µMT mice at 4 h and again at 8 days postinfection (p.i.). Serum transfer was delayed until 4 h p.i. to allow for clearance of amastigotes from the circulation (15) and to obviate any immediate opsonic effects of antileishmanial Ab (16).
Neutrophil depletion
Monoclonal rat anti-mouse granulocyte (RB6-8C5; Ref. 17), a generous gift from Dr. R. Coffman (DNAX, Palo Alto, CA) and Dr. S. Reiner (University of Chicago, Chicago, IL), was purified from cell culture supernatant by affinity chromatography using a protein G column. RB6-8C5 mAb (0.2 mg) or control rat IgG (Sigma) was administered i.p. at day -1 of infection and every third day thereafter. The efficacy of depletion was monitored by flow cytometric analysis of spleen cells. Control naive µMT, C57BL/6, and BALB/c mice were also treated with RB6-8C5 and observed daily over the time course of infections. Neutrophil depletion did not lead to any deterioration in health status in naive mice. However, we did note a slight reduction in the frequency of peripheral (splenic) CD8+ T cells in both naive and infected RB6-8C5-treated mice (data not shown).
Immunohistology
Neutrophils were identified by staining with RB6-8C5 using the Vectastain Elite immunoperoxidase system (Vector Laboratories, Peterborough, U.K.). Briefly, livers were collected from mice killed by cervical dislocation, immediately embedded in OCT compound (Raymond Lamb, London, U.K.), and snap frozen in isopentane/liquid nitrogen. Sections (6 µm) were cut with a cryostat and were fixed for 2 min in acetone, air dried, and stored at -20°C until stained. Frozen sections were fixed for 8 min in acetone and washed for 20 min in PBS (10 mM NaH2PO4, pH 7.5, and 0.9% (w/v) saline) immediately before staining. Sections were additionally blocked with avidin for 15 min, and then with biotin for 15 min followed by 1.5% (v/v) rabbit serum diluted in PBS for 30 min. Excess serum was blotted from sections that were then incubated with primary Ab, diluted in PBS, for 30 min. RB6-8C5 and control rat IgG were used at 2.5 µg/ml. Abs were detected by incubation with biotinylated rabbit-anti-rat IgG (mouse adsorbed; 1:100 (v/v) dilution), and this was subsequently detected with avidin biotinylated-HRP complexes. Sections were developed for peroxidase activity in 3,3-diaminobenzidine tetrahydrochloride (DAB) developing substrate. Sections were counterstained for 1 min with Harris hematoxylin, dehydrated and mounted in DePeX (BDH, Poole, U.K.).
Granuloma counting
Liver sections from infected mice were stained with hematoxylin
and eosin and the degree of the granulomatous responses assessed in two
ways: 1) granuloma density was determined from 
150 fields of view
per mouse liver (x63 magnification; n = 2–3
mice/group) and 2) the degree of maturation of granulomas was scored
around infected Kupffer cells (KC), as described elsewhere
(18).
Statistical analysis
Data were analyzed using either the nonpaired Students
t test, Peasons linear correlation, or
2 as appropriate, using the graphics package
Fig P (Biosoft, Cambridge, U.K.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Comparisons of the course of L. donovani infection in
age- and sex-matched µMT and C57BL/6 mice revealed that µMT mice
were highly resistant to infection (Fig. 1
). Wild-type C57BL/6 mice displayed
organ-specific control of L. donovani infection, as
previously reported in BALB/c mice (5, 19). Hence, liver
parasite loads began to resolve 21–28 days after infection, whereas
parasites persisted indefinitely in the spleen, albeit at lower levels
than in the liver. Not only were peak parasite loads in both organs
significantly lower in µMT mice, (Fig. 1; p < 0.05
at day 28 for liver; p < 0.005 at day 56 for spleen)
but the long-term outcome of infection was dramatically altered.
Hepatic infection in the µMT mouse was reduced to below the limits of
detection by impression smears within 8 wk of infection. Furthermore,
µMT mice did not develop persistent infection in the spleen (Fig. 1).
Hence, B cell-deficient µMT mice are highly resistant to primary
infection with L. donovani.
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). In C57BL/6 mice, hepatomegaly was
greatest following peak parasite load (1.91 ± 0.25-fold increase
compared with naive at day 36 p.i.; p < 0.001)
and at the time when T cell-dependent immune mechanisms started to
reduce parasite numbers. In contrast, peak hepatomegaly in µMT mice
preceded peak parasite burden (1.31 ± 0.34-fold increase compared
with naive at day 14 p.i.; p < 0.002), returning
to control levels as parasites were cleared from the tissue. However,
overt liver pathology was also noted in µMT mice at this time (see
Fig. 3 and below). Splenomegaly was a
dominant feature of late infection in C57BL/6 mice (10.22 ±
0.31-fold increase compared with naive at day 56 p.i.;
p < 0.0001; Fig. 2), reflecting the lack of clearance
of parasites from this organ. In contrast, although µMT mice
demonstrated more rapid onset of splenomegaly (6.61 ± 1.52-fold
vs a 1.71 ± 0.23-fold increase in C57BL/6 mice at day 14
p.i.; p < 0.05), this was self-limiting in µMT mice,
again commensurate with the reduction in parasite load in this
organ.
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L. donovani infection causes a structured hepatic
tissue response in most mouse strains, in which infiltrating monocytes,
lymphocytes, and neutrophils aggregate around infected KC to form
granulomas (1, 2). The kinetics of this response in
wild-type and µMT mice was very different however, as shown in Fig. 4A. At day 14 p.i., the
livers of µMT mice contained more than three times the number of
granulomas, compared with wild-type mice (p <
0.001). Furthermore, unlike in wild-type mice where granulomas
persisted throughout the period of parasite clearance, the granulomas
of µMT mice were transient and these structures rapidly disappeared
from the tissue as parasites were eliminated.
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60% of infected KC (Fig. 4
B). In contrast, 90% of
infected KC in µMT mice had elicited a local inflammatory response by
this time point. Two additional features of the response in µMT mice
were also evident from this analysis. First, µMT mice were found to
contain many "empty" granulomas at day 14 p.i. (excluded from
the analysis shown in Fig. 4B). These most likely
represented granulomas in which parasites had already been eliminated,
and were only rarely observed in C57BL/6 mice at this time. Second,
although parasites within some granulomas were being cleared by day
14 p.i., few granulomas had reached a mature stage of development
(defined by an organized cuffing of infiltrating cells). Hence, in the
absence of B cells, there are marked changes in the intensity and
kinetics of the hepatic granulomatous response to L.
donovani. Increased presence of neutrophils in the liver and spleen of µMT mice following infection with L. donovani
Between day 14 and day 21 p.i., the livers of µMT mice
exhibited signs of gross destructive pathology (Fig. 3). They were pale
in color, suggesting occlusion of blood vessels, and large areas of
necrosis were frequent in hematoxylin and eosin-stained sections (data
not shown). Neutrophils, stained with mAb RB6-8C5, were commonly
associated with these areas of tissue damage, suggesting the latter was
the result of an excessive inflammatory response. In contrast, liver
necrosis was not observed in infected C57BL/6 mice at any time p.i.
Although mononuclear cells were still abundant in the granulomas of
µMT mice, neutrophils were also readily detected, suggesting the
presence of continued active inflammation within these granulomas (Fig. 5). In contrast, neutrophils were scarce
in the granulomas of C57BL/6 mice infected with L. donovani
(Fig. 5 and Refs. 2 and 20).
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Neutrophil depletion leads to enhanced parasite growth in B cell deficient and wild-type mice
The increased presence of neutrophils in the liver granulomas of
µMT mice and their presence near regions of hepatic necrosis
suggested that these cells may contribute both to the pathological
level of inflammation, as well as the accelerated clearance of
parasites observed in µMT mice. To address these questions, µMT and
C57BL/6 mice were treated with the neutrophil-depleting mAb RB6-8C5
(21, 22). Neutrophil depletion over the first 2 wk of
infection had a surprising and dramatic effect on parasite development
in both strains of mice (Table I).
Parasite burden was increased 6-fold in the spleen and liver of C57BL/6
mice, resulting in parasite burdens in the latter organ that are not
approached even at the peak of disease progression in untreated mice
(see Fig. 1). RB6-8C5 treatment also increased parasite loads in µMT
mice, although to a greater relative extent than seen in C57BL/6 mice.
This may reflect a more critical role for neutrophils in µMT mice or
their greater relative abundance.
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Serum transfer abrogates gross pathology, but minimally affects the resistance of µMT mice to L. donovani infection
Other researchers (23, 24) have previously implicated serum Ig and/or immune complexes in the regulation of anti-inflammatory cytokine responses. Therefore, we evaluated whether the lack of serum Ig, a consequence of the B cell deficiency in µMT mice, had any immunoregulatory consequences in our infection model.
To address this question, µMT mice were infected with L.
donovani and at 4 h p.i. (a time when >95% of parasites
were cleared from the circulation (15)); we then
adoptively transferred 200 µl of serum derived from naive C57BL/6
mice or mice infected for 56 days with L. donovani. Serum
transfer was repeated at day 8 p.i., and mice were sacrificed at
day 16 p.i. The results of this experiment were both striking and
unexpected. Transfer of either normal or chronic-infection serum had
minimal effect on the resistance of µMT mice to infection with
L. donovani (Fig. 6).
Furthermore, immunohistological analysis using RB6-8C5 demonstrated
that the levels of active inflammation (i.e., neutrophil infiltration)
within the granulomas of control and serum-treated µMT mice was
similar (data not shown). However, serum transfer almost totally
inhibited the gross destructive pathology observed in the livers of
µMT mice. Whereas 5/5 control µMT mice showed clear outward signs
of liver necrosis (as illustrated in Fig. 3B), this was seen
in only 1/5 µMT mice receiving normal mouse serum and in 0/5 mice
receiving chronic-infection serum. All other serum recipients had
livers with normal gross appearance. Hematoxylin and eosin staining of
tissue section also revealed few or no signs of necrosis in serum
recipients (data not shown). Hence, serum transfer distinguishes the
mechanisms involved in the development of tissue damage and enhanced
host resistance in µMT mice.
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Although serum reconstitution protects µMT mice from tissue
damage, it had minimal effect on the enhanced levels of resistance of
µMT compared with wild-type mice. We therefore sought other
explanations for the heightened resistance of µMT mice to L.
donovani. As lack of B cells has been reported to have effects on
the function of T cells in other systems, we wished to determine
whether T cells in µMT mice and C57BL/6 mice differed in their
ability to protect against L. donovani infection. Previous
studies have illustrated the importance of both
CD4+ and CD8+ T cells for
immunity to L. donovani in the liver (3).
Hence, we copurified both populations directly by MACS using a
combination of anti-CD4 and anti-CD8 mAbs. Purified T cells
from C57BL/6 mice were 74% CD4+ and 28%
CD8+, whereas those isolated from µMT mice were
76% CD4+ and 23% CD8+.
These mixed populations of T cells (104 or
105) were adoptively transferred to C57BL/6
background RAG1-/- mice 1 day before infection
with L. donovani. On day 28 p.i., mice were sacrificed
and each mouse analyzed individually for parasite load and the degree
of CD4+ and CD8+ T cell
reconstitution (Fig. 7). The data clearly
demonstrate that control of L. donovani in this transfer
model was correlated with CD4+
(p < 0.002 and p < 0.05 for
µMT and C57BL/6 donors, respectively) rather than
CD8+ T cell number in reconstituted mice. More
importantly, there was no difference in the ability of
CD4+ or CD8+ T cells from
C57BL/6 and µMT mice to transfer protection to
RAG1-/- mice. Hence, the development of T cells
in a B cell-deficient environment does not result in any quantitative
alterations in their ability to transfer protection against L.
donovani infection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The rapid curing response of µMT mice following L. donovani infection is striking in a number of respects. First, the rapid clearance of parasites in the liver of µMT mice extends both the rate of cure and the degree of pathology beyond those previously defined using MHC congenic mice on the C57 background (25). This is associated with a rapid granulomatous response, whose cellular composition is neutrophil-rich relative to wild-type mice. This suggests the continuation of a process of active inflammation in these granulomas, though this does not culminate in necrosis at these sites. These granulomas effectively eliminate parasites, but without the maturation associated with parasite clearance in wild-type mice. Indeed, unlike the response in C57BL/6 mice and other strains (this manuscript and Refs. 2 and 12), the granulomas in µMT mice are transient, their numbers declining rapidly after parasite clearance. Second, µMT mice are able to effectively control parasites within the spleen, and fail to develop long-lasting splenomegaly and persistent parasite load characteristic of wild-type C57BL/6 mice and BALB/c mice (this manuscript and Ref. 5). The lack of persistence of parasites in the spleen of µMT mice illustrates that immunological effector mechanisms are able to achieve effective control of parasites in this organ, and that these are uncovered in the absence of B cells.
Neutrophil depletion led to dramatic increases in parasite growth compared with that seen in untreated controls, in both µMT and also in C57BL/6 and BALB/c mice. Although the relative increase in parasite burden was slightly greater in µMT mice compared with wild-type mice, this did not appear to fully reflect the observed differences in the numbers of neutrophils within the granulomas of these mice. A possible explanation for this discrepancy is that parasite density in the infected organ becomes a limiting factor. This contention is supported by the observation that in mice with very low levels of infection (liver LDU < 100), the effect of neutrophil depletion is significantly more marked in µMT mice than in C57BL/6 mice (S. C. Smelt, unpublished observations). Nevertheless, these data clearly indicate that in normal mice, as well as in B cell-deficient mice, neutrophils play a vital, but hitherto unrecognized role in the control of L. donovani infection.
The direct demonstration of a role for neutrophils in the control of
L. donovani infection was unexpected, particularly in
wild-type C57BL/6 and BALB/c mice. A small number of neutrophils can be
detected early after infection infiltrating the liver parenchyma and
subsequently within the granuloma (Fig. 5 and Refs. 2 and
19); nevertheless, the granuloma is usually predominantly
mononuclear in composition. Clearly, even the limited numbers of
neutrophils seen in the tissue are able to make a substantial
contribution to host resistance. A balanced neutrophil response may, at
the expense of maximal anti-leishmanial activity, represent a
mechanism for protecting against excess pathology. In this regard, we
have recently shown that anti-CTLA-4 treatment of mice also
enhances granuloma formation and parasite clearance. However, unlike
the situation seen in µMT mice, the response of
anti-CTLA-4-treated mice remains predominantly mononuclear and
progresses rapidly to full maturation (18). Furthermore,
the enhanced clearance of parasites induced by anti-CTLA-4 does not
result in any overt liver destruction. Comparison of these two
experimental manipulations of the host response to L.
donovani supports the notion that successful elimination of
pathogens and the containment of pathology requires an appropriately
balanced cellular response.
Recent data suggest at least one mechanism by which neutrophils may
contribute to the control of L. donovani infection. Studies
with gene targeted gp91 (phox-/-)
and NOS2-/- mice, have demonstrated that while
late stages of disease resolution are controlled exclusively by
reactive nitrogen intermediates (RNI), reactive oxygen intermediates
(ROI) play an important role in the early regulation of parasite
multiplication and mononuclear cell recruitment (20).
Although these ROI have been assumed to derive from monocytes or
macrophages, our data would suggest that neutrophils may make an
important, if not exclusive, contribution to ROI production. The
production of ROI by neutrophils is regulated by TNF-
, and we have
already shown that TNF- producing cells are recruited into the liver
within 3 days of infection with L. donovani
(19). In contrast, infected KC fail to make TNF- at
this early stage, and have also been shown to be relatively poor
producers of ROI (26). Thus, depletion of neutrophils may
limit this early effector response and promote early parasite
multiplication in KC. However, the increase in parasite loads we have
observed in neutrophil-depleted mice are significantly greater than
that seen in phox-/- mice, suggesting
other roles for neutrophils in the early response. These may include
direct parasite killing or the liberation of cytokines which directly
or indirectly amplify the host response. Neutrophils are capable of
producing a number of cytokines, including TNF-, IL-1 L-1ß,
macrophage-inflammatory protein-2, transforming growth factor-ß1,
macrophage-inflammatory protein-1, IL-10, and IL-12.
(27, 28, 29, 30). Many of these cytokines/chemokines have already
been shown to play important roles in L. donovani infection
(1, 19, 31, 32, 33). Further work involving neutrophil
depletion in cytokine gene targeted mice will be required to clarify
this issue. Unfortunately, effective neutrophil elimination for greater
than 14 days was not possible, due to increasingly rapid replenishment
of the neutrophil pool (S. C. Smelt, unpublished observations),
and thus it remains to be determined whether in the absence of
neutrophils, mice retain the capacity to eventually resolve their
infections. The latter might be predicted by a switch to a dependency
on macrophage-derived RNI within the granulomas (20).
Although there have been a number of immunological aberrations reported in µMT mice, to our knowledge this is the first report of an exaggeration in neutrophil function. IL-10 has been reported as a major regulator of neutrophil function, and B cells have been reported to produce IL-10 in a number of infectious disease settings (34, 35, 36). We have detected IL-10 mRNA in B cells isolated from infected C57BL/6 and BALB/c mice (S. C. Smelt, C. R. Engwerda, and P. M. Kaye, unpublished data), but as this occurs relatively late in infection, we believe it unlikely that B cell-derived IL-10 contributes to the negative regulation of neutrophil function early in infection. Attempts to revert the phenotype of µMT mice by the adoptive transfer of B cells from C57BL/6 mice have been inconclusive, as like others (Ref. 37 ; D. van Essen and D. Gray, unpublished observations), we have only been able to achieve reconstitution levels of <10%. At this level, we have not observed any significant differences in parasite burdens or pathology between unreconstituted and B cell-reconstituted µMT mice (data not shown).
In contrast to our inability to modulate the response of µMT mice by B cell transfer, transfer of either normal or chronic-infection serum has a profound but highly selective effect in these mice. Serum transfer has minimal or no effect on parasite load in µMT mice indicating that the enhanced resistance of µMT mice is independent of the presence or absence of either normal or parasite specific Ig. However, µMT mice reconstituted with either normal or chronic-infection serum showed no signs of hepatic destruction. These data suggest that the presence of Igs, and possibly immune complexes (38) result in protection from tissue destructive processes. These data also highlight that the mechanisms controlling destructive pathology are independent of those which regulate the heightened resistance of µMT mice to L. donovani infection. Ig has also been shown to be required for the down-modulation of the schistosome egg granuloma, and in this model too, there is a dissociation between the impact on pathology and host resistance (23). However, whereas in schistosomiasis pathology remains focal around deposited eggs, such an association between the site of tissue destruction and the presence of the triggering stimulus is less easy to confirm in the case of L. donovani infection. Importantly, L. donovani infection of µMT mice now provides an additional tool for future studies into the mechanisms which control these two distinct host processes.
Although dysregulation of the neutrophil response is the most evident phenotype in µMT mice infected with L. donovani, our data do not formally rule out other more subtle influences on effector function. Indeed, the maintenance of a highly resistant phenotype even after serum transfer and the elimination of pathology suggests this is the case. The impact of B cells on the development and function of T cells has been extensively analyzed, though often with conflicting results. In L. major infections, the presence or absence of B cells fails to influence the commitment to Th 1 or Th 2 development (11). Similarly, immunization with acetylcholine receptor in CFA produces identical responses in wild-type and µMT mice (39), as was the case in a murine model for allergic asthma (40). In contrast, there have been numerous examples where the development of T cell responses following infection is modulated by the absence of B cells. µMT mice are defective in switching from a Th1 to a Th2 response following Plasmodium chabaudi chabaudi infection (41). Similarly, after infection with Chlamydia, µMT mice fail to mount a significant delayed-type hypersensitivity response and have higher mortality rates than their wild-type counterparts (42). Furthermore, µMT mice demonstrate more rapid graft rejection (43), enhanced primary LCMV-specific CTL responses at high viral loads (38), and a higher death rate among activated CD8+ cells and LCMV-specific CD8+ CTL memory cells after virus infection (44). In our hands, T cells derived from either strain have equal protective efficacy on a cell per cell basis when transferred into RAG1-/- mice. These data confirm that T cells originating from an environment with or without B cells can be primed in the absence of B cells and can develop the immune phenotype required for efficient clearance of L. donovani. These data do not argue for or against a subsequent role of B cells in Ag presentation or cytokine regulation of T cell immunity. In addition, the striking rapidity with which hepatic granulomas are lost from the tissue after parasite clearance may suggest a role for B cells in T cell persistence within granulomas. Studies are in progress to address these important issues.
In summary, this study demonstrates that µMT mice are highly resistant to L. donovani infection, but suffer from excessive destructive pathology. These two features of infection in µMT mice can be dissociated by the transfer of serum Ab, which limits pathology without compromising host resistance. Furthermore, we have shown that neutrophils are an essential component of host resistance in wild-type mice, and that their role is exaggerated in the absence of B cells. These data suggest a hitherto unrecognized negative association between B cells and neutrophils in the host response to infection.
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Acknowledgments |
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Footnotes |
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2 Current address: IMM6, Department of Neuropharmacology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.
3 Address correspondence and reprint requests to Dr. P. M. Kaye, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K. E-mail address:
4 Abbreviations used in this paper: VL, visceral leishmaniasis; KC, Kupffer cell; LDU, Leishman Donovan units; µMT, IgM transmembrane domain; RAG1-/-, recombinase activating gene 1; p.i., postinfection.
Received for publication July 19, 1999. Accepted for publication January 31, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor type I. J. Exp. Med. 188:217. in experimental visceral leishmaniasis. J. Immunol. 153:768.[Abstract]
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A. Mizoguchi and A. K. Bhan A Case for Regulatory B Cells J. Immunol., January 15, 2006; 176(2): 705 - 710. [Abstract] [Full Text] [PDF]
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B. Dondji, E. Perez-Jimenez, K. Goldsmith-Pestana, M. Esteban, and D. McMahon-Pratt Heterologous Prime-Boost Vaccination with the LACK Antigen Protects against Murine Visceral Leishmaniasis Infect. Immun., August 1, 2005; 73(8): 5286 - 5289. [Abstract] [Full Text] [PDF]
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A. Casadevall and L.-a. Pirofski New Concepts in Antibody-Mediated Immunity Infect. Immun., November 1, 2004; 72(11): 6191 - 6196. [Full Text] [PDF]
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C. P. Simmons, S. Clare, M. Ghaem-Maghami, T. K. Uren, J. Rankin, A. Huett, R. Goldin, D. J. Lewis, T. T. MacDonald, R. A. Strugnell, et al. Central Role for B Lymphocytes and CD4+ T Cells in Immunity to Infection by the Attaching and Effacing Pathogen Citrobacter rodentium Infect. Immun., September 1, 2003; 71(9): 5077 - 5086. [Abstract] [Full Text] [PDF]
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 J. A. R. Hobbs, R. May, K. Tanousis, E. McNeill, M. Mathies, C. Gebhardt, R. Henderson, M. J. Robinson, and N. Hogg Myeloid Cell Function in MRP-14 (S100A9) Null Mice Mol. Cell. Biol., April 1, 2003; 23(7): 2564 - 2576. [Abstract] [Full Text] [PDF]
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H. Shen, J. K. Whitmire, X. Fan, D. J. Shedlock, S. M. Kaech, and R. Ahmed A Specific Role for B Cells in the Generation of CD8 T Cell Memory by Recombinant Listeria monocytogenes J. Immunol., February 1, 2003; 170(3): 1443 - 1451. [Abstract] [Full Text] [PDF]
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M. Colmenares, S. L. Constant, P. E. Kima, and D. McMahon-Pratt Leishmania pifanoi Pathogenesis: Selective Lack of a Local Cutaneous Response in the Absence of Circulating Antibody Infect. Immun., December 1, 2002; 70(12): 6597 - 6605. [Abstract] [Full Text] [PDF]
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A. J. Buendia, L. Del Rio, N. Ortega, J. Sanchez, M. C. Gallego, M. R. Caro, J. A. Navarro, F. Cuello, and J. Salinas B-Cell-Deficient Mice Show an Exacerbated Inflammatory Response in a Model of Chlamydophila abortus Infection Infect. Immun., December 1, 2002; 70(12): 6911 - 6918. [Abstract] [Full Text] [PDF]
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K. Venuprasad, P. P. Banerjee, S. Chattopadhyay, S. Sharma, S. Pal, P. B. Parab, D. Mitra, and B. Saha Human Neutrophil-Expressed CD28 Interacts with Macrophage B7 to Induce Phosphatidylinositol 3-Kinase-Dependent IFN-{gamma} Secretion and Restriction of Leishmania Growth J. Immunol., July 15, 2002; 169(2): 920 - 928. [Abstract] [Full Text] [PDF]
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J. G. Valenzuela, Y. Belkaid, M. K. Garfield, S. Mendez, S. Kamhawi, E. D. Rowton, D. L. Sacks, and J. M.C. Ribeiro Toward a Defined Anti-Leishmania Vaccine Targeting Vector Antigens: Characterization of a Protective Salivary Protein J. Exp. Med., August 6, 2001; 194(3): 331 - 342. [Abstract] [Full Text] [PDF]
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M. M. Kane and D. M. Mosser The Role of IL-10 in Promoting Disease Progression in Leishmaniasis J. Immunol., January 15, 2001; 166(2): 1141 - 1147. [Abstract] [Full Text] [PDF]
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C. M. Bosio and K. L. Elkins Susceptibility to Secondary Francisella tularensis Live Vaccine Strain Infection in B-Cell-Deficient Mice Is Associated with Neutrophilia but Not with Defects in Specific T-Cell-Mediated Immunity Infect. Immun., January 1, 2001; 69(1): 194 - 203. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and C57BL/6 (
) mice were infected with 2 x
107 L. donovani amastigotes, and parasites
loads were determined in the liver (A) and spleen
(B) at the times indicated. Data represent mean LDU
± SE (n = 3 mice) and are representative of three
independent experiments with similar results. Significance levels
comparing µMT and C57BL/6 mice are indicated; *,
p < 0.05; **, p <
0.005.
) and
µMT (
) mice. Data were obtained by examining 
)
mice were cotransferred to RAG1-/- mice 1 day before
infection with L. donovani. Control
RAG1-/- mice received no cells (*). At day 28
p.i., parasite load was determined from impression smears and the
frequency of CD4+ and CD8+ T cells in the
spleen determined by flow cytometry. Data represent hepatic parasite
load (LDU) for individual mice, plotted against either CD4+
(A) or CD8+ (B) T cell
frequency. A significant correlation was observed for CD4+
cells derived from either µMT (r = -0.86,
p < 0.002) or C57BL/6 (r =
-0.84, p < 0.005) donors, whereas no significant
correlations were seen for CD8+ cells of either
strain.