Regional differences in the cellular immune response to experimental cutaneous or visceral infection with Leishmania donovani

Abstract

Infection with the protozoan Leishmania donovani can cause serious visceral disease or subclinical infection in humans. To better understand the pathogenesis of this dichotomy, we have investigated the host cellular immune response to cutaneous or visceral infection in a murine model. Mice infected in the skin developed no detectable visceral parasitism, whereas intravenous inoculation resulted in hepatosplenomegaly and an increasing visceral parasite burden. Spleen cells from mice with locally controlled cutaneous infection showed strong parasite-specific proliferative and gamma interferon (IFN-gamma) responses, but spleen cells from systemically infected mice were unresponsive to parasite antigens. The in situ expression of IFN-gamma, interleukin-4 (IL-4), IL-10, IL-12, and inducible nitric oxide synthase (iNOS) mRNAs was determined in the spleen, draining lymph node (LN), and cutaneous site of inoculation. There was considerably greater expression of IFN-gamma and IL-12 p40 mRNAs in the LN draining a locally controlled cutaneous infection than in the spleen following systemic infection. Similarly, there was a high level of IFN-gamma production by LN cells following subcutaneous infection but no IFN-gamma production by spleen cells following systemic infection. Splenic IL-4 expression was transiently increased early after systemic infection, but splenic IL-10 transcripts increased throughout the course of visceral infection. IL-4 and IL-10 mRNAs were also increased in the LN following cutaneous infection. iNOS mRNA was detected earlier in the LN draining a cutaneous site of infection compared to the spleen following systemic challenge. Thus, locally controlled cutaneous infection was associated with antigen-specific spleen cell responsiveness and markedly increased levels of IFN-gamma, IL-12, and iNOS mRNA in the draining LN. Progressive splenic parasitism was associated with an early IL-4 response, markedly increased IL-10 but minimal IL-12 expression, and delayed expression of iNOS.

FIG. 1

Kinetics of tissue parasite burden in L. donovani-infected mice. Mice were infected with 5 × 10⁶ L. donovani amastigotes by either s.c. or i.v. inoculation. Spleens and/or lymph nodes were harvested at 2, 7, 14, 28, 42, and 56 days after infection, footpad skin was harvested at 7, 14, 28, and 42 days after infection, and the total organ parasite burden was quantified by limiting dilution culture. The tissue parasite burden is expressed as the log of the reciprocal of the highest culture-positive dilution (mean of five samples and standard error [bars]). Results are shown for the parasite burden per milligram of tissue or for the total organ. The parasite burden in the skin was determined only per milligram of tissue because excision of the entire footpad was difficult. The spleens of the s.c.-infected mice were culture negative (<1 parasite/mg of tissue) at all time points. The differences between the parasite burden per milligram of spleen and LN tissue was statistically significant at days 14, 28, and 56 (P = 0.03, P = 0.006, and P = 0.01, respectively).

FIG. 2

Proliferative response of spleen cells from L. donovani-infected mice. Spleen cells were obtained from s.c.- and i.v.-infected mice and uninfected age-matched controls on days 7, 14, 28, 42, and 56 after infection. Cells were cultured in quadruplicate wells and stimulated for 3 days with ConA (5 μg/ml) or medium alone (left) or for 5 days with SLDA (25 μg/ml) or medium alone (right). One microcurie of [³H]thymidine was added to the cultures for the last 16 to 18 h, and incorporation was determined by scintillation counting. Results are expressed as the mean and standard error (bars) of the stimulation indices (mean counts per minute of stimulated wells/mean counts per minute of unstimulated wells) of groups of four mice except for the antigen-induced proliferation of day 28 s.c.-infected mice, where data from only one mouse were available.

FIG. 3

IFN-γ production by spleen (left) and LN (right) cells from L. donovani-infected mice. Cell cultures were set up as described for Fig. 2. Supernatants from SLDA-stimulated and unstimulated (medium control) spleen cells were collected after 5 days culture and assayed for IFN-γ by sandwich ELISA. Results shown are from uninfected controls and from s.c.- and i.v.-infected mice and are expressed as the mean and standard error (bars) of the fold increase in IFN-γ production of SLDA-induced over unstimulated cells. Results are from quadruplicate cultures from one to four mice per group. External sacral LN cells from uninfected mice were not studied.

FIG. 4

Ex vivo IFN-γ secretion by LN and spleen cells isolated from L. donovani-infected and uninfected mice. Popliteal LN cells from s.c.-infected mice and spleen cells from i.v.-infected mice, or LN and spleen cells from control uninfected mice, were cultured as described for Fig. 2 in the absence of exogenous antigen stimulation. Supernatants were harvested on day 5 of culture and assayed for IFN-γ by sandwich ELISA. Results are expressed as the mean and standard error (bars) of quadruplicate cultures of two to four mice per group.

FIG. 5

Representative standard curves used for quantitation of the RT-PCR amplification products. Fivefold serial dilutions of purified cytokine and HPRT cDNA templates were amplified by using specific primers, and the product was quantified by fluid-phase hybridization with a labeled internal oligonucleotide probe and colorimetric detection. Shown are the linear portions of the curves which were used for interpolation in the determination of the quantity of RT-PCR-amplified mRNA from tissue samples. The values are graphed as the optical density at 405 nm (OD₄₀₅) of the colorimetric reaction versus the concentration of the input purified cDNA template.

FIG. 6

Splenic cytokine expression in L. donovani-infected and uninfected mice. Mice were infected with 5 × 10⁶ L. donovani amastigotes by either s.c. or i.v. inoculation. Age-matched uninfected mice served as controls. Spleens were harvested at 2, 7, 14, 28, and 56 days after infection, and total RNA was isolated. Then 0.5 μg of total RNA was pooled from the spleens of five mice in each group, reverse transcribed, amplified by PCR, and quantified by fluid-phase hybridization with a labeled internal oligonucleotide probe and colorimetric detection (RT-PCR ELISA). For each sample, the quantity of input cDNA was interpolated from the standard curve. Minor differences in the quantity of total RNA between samples were corrected by normalization to the quantity of HPRT in the same sample. The quantity is expressed as the femtograms of cytokine mRNA normalized to the quantity of HPRT mRNA.

FIG. 7

Popliteal LN cytokine expression in L. donovani-infected mice. Mice were infected s.c. in the footpad and the draining popliteal LN harvested. Age-matched uninfected mice served as controls. Total RNA was isolated and the cytokine and HPRT mRNAs quantified by RT-PCR ELISA, and results are presented as described for Fig. 6.

FIG. 8

Splenic and LN expression of iNOS mRNA in L. donovani-infected and uninfected mice. Mice were infected s.c. or i.v. or received HBSS alone by the same route. The draining popliteal LN and spleens were harvested as described in the legend to Fig. 6. Total RNA was isolated, and the iNOS and HPRT mRNAs were analyzed by Southern blotting (lower panels) and quantified by RT-PCR ELISA (top panels). Lanes 1 to 4 on the Southern blot correspond to 0.8, 0.16, 0.032, and 0 fg of input purified HPRT or iNOS cDNA template, respectively. Other lanes: I, i.v.; S, s.c.; C, uninfected control. The iNOS mRNA quantified by RT-PCR ELISA is expressed as the femtograms of iNOS mRNA normalized to the quantity of HPRT mRNA. The results are presented as described in the legend to Fig. 6.