IL-12p40 is produced rapidly and transiently in the spleen by CD8⁺ DC following <i>L. donovani</i> infection.

<p>(A) Mice were infected with 2×10⁷<i>L. donovani</i> amastigotes i.v. and sacrificed at 2 hr, 5 hr, 12 hr, 24 hr, 72 hr, 5 days and 7 days p.i. mRNA was extracted from the spleen (squares), liver (inverted triangles) and bone marrow (triangles) at each time point and accumulation of IL-12p40 mRNA was detected by real-time RT-PCR and is expressed as mRNA molecule per 1000 HPRT molecules. Data are representative of 3 mice per time point. (B) Mice were infected with 1×10⁸<i>L. donovani</i> amastigotes i.v. and spleens were removed at 5 hr p.i. MACS-enriched DC were enumerated by labelling with anti-CD11c mAb and examined for expression of isotype control mAb or IL-12p40 (1,000,000 events collected, 10,000 events shown). Cells were then electronically gated, based on expression of IL-12p40, as indicated, and then examined for expression of CD4 and CD8 (5000 events shown). Gates were determined from an isotype control for IL-12p40, in which 0.8% of CD11c⁺ cells were shown to be positive. Numbers below rectangular gates indicate the percentage of cells in that gate, while numbers in the upper right hand corners of FACS profiles indicate the percentage of cells in the indicated quadrants. One representative experiment of three performed with similar outcome is shown (n = 4 mice per treatment group in each experiment).</p

VCAM-1 is required for efficient control of <i>L. donovani</i> infection in the liver.

<p>Female C57BL/6 mice were treated with anti-VCAM-1 mAb (closed bars) or control rat IgG (open bars) and infected with 2×10⁷<i>L. donovani</i> amastigotes i.v. Mice were injected i.p. with 1 mg of antibody prior to infection and every 3 days thereafter. Parasite burdens were determined in the liver at day 14 p.i. (A) and data represent the mean±SEM in Leishman Donovan units. The number and maturity of hepatic granulomas were estimated on day 14 liver sections stained with anti-<i>L. donovani</i> sera (B). Data represent the frequency of infected Kupffer cells (KC), immature granulomas (IG) and mature granulomas (MG) per liver. Hepatic mononuclear cells were isolated from naïve (hatched bars) and infected mice on day 14 p.i. and enumerated (C). mRNA was extracted from livers of naïve or day 14 p.i. VCAM-1 blocked or control mice, and accumulation of IFNγ (D), TNF (E) and NOS-2 (F) mRNA was detected by real-time RT-PCR and is expressed as mRNA molecule per 1000 HPRT molecules. One representative experiment of two performed with similar outcome is shown (n = 4 mice per treatment group in each experiment). Statistical differences of p≤0.05 (*) or p<0.01 (**) for control versus anti-VCAM-1 treatment are indicated.</p

VCAM-1 expression in the spleens of naive C57BL/6 mice.

<p>(A) VCAM-1 localisation (green) in a tissue section from a naïve mouse. Marginal metallophilic macrophages were stained with MOMA-1 mAb (red) allowing visualisation of red pulp (RP), white pulp (WP) and marginal zone (MZ) regions of the spleen, as indicated (×100) The tissue section was mounted in media containing DAPI to stain cell nuclei (blue). (B-D) Staining for VCAM-1 expression, red pulp macrophages (F4/80⁺), MM macrophages (MOMA-1⁺), MZ macrophages (ERTR9/SIGNR1⁺), MZ sinus-lining endothelial cells (Meca-32⁺) and DC (CD11c⁺) (×630) is shown in colours indicated above panels. White arrows in (C) and (D) indicate areas where VCAM-1⁺ cells (yellow) are in close proximity to DC (blue).</p

VLA-4 is required for efficient control of <i>L. donovani</i> infection in the liver.

<p>Female C57BL/6 mice were treated with anti-VLA-4 mAb (closed bars) or control rat IgG (open bars) and infected with 2×10⁷<i>L. donovani</i> amastigotes i.v. Mice were injected i.p. with 1 mg of antibody prior to infection and every 3 days thereafter. Parasite burdens were determined in the liver at day 14 p.i. (A) and data represent the mean±SEM in Leishman Donovan units. The number and maturity of hepatic granulomas was estimated on day 14 liver sections stained with anti-<i>L. donovani</i> sera (B). Data represent the frequency of infected Kupffer cells (KC), immature granulomas (IG) and mature granulomas (MG) per liver. Hepatic mononuclear cells were isolated from naïve (hatched bars) and infected mice on day 14 p.i. and enumerated (C). mRNA was extracted from livers of naïve (hatched bars) or day 14 p.i. VLA-4 blocked (closed bars) or control mice (open bars), and accumulation of IFNγ (D), TNF (E) and NOS-2 (F) mRNA was detected by real-time RT-PCR and is expressed as mRNA molecule per 1000 HPRT molecules. One representative experiment of two performed with similar outcome is shown (n = 4 mice per treatment group in each experiment). Statistical differences of p<0.05 (*) or p<0.01 (**) for control versus anti-VLA-4 treatment are indicated.</p

VCAM-1/VLA-4 interactions are not critical for DC migration into the spleen.

<p>Naïve C57BL/6 mice were injected with 100 µg FITC-dextran i.v. followed by 1 mg of control rat IgG (A) or anti-VCAM-1 mAb (B) i.p. 24 hr later. Hoechst 33342-labelled splenic CD11c⁺ DC (1×10⁶) were administered i.v. 1 hr following mAb injection. The following day mice were either left as naïve or infected with 2×10⁷<i>L. donovani</i> amastigotes i.v. and spleens were removed 5 hr later (24 hr post-cell transfer). The distribution of Hoechst 33342-labelled cells was analysed in 20 µm sections and photographed under UV illumination (×100). Data are representative of one of two experiments performed (n = 3 mice per group). The number of Hoechst 33342-labelled cells was determined from 15 fields of view per mouse spleen (×25 magnification) (C).</p

VCAM-1/VLA-4 interactions are not required for leukocyte migration into the liver following <i>L. donovani</i> infection.

<p>Female C57BL/6 mice were untreated (vertical hatched bar) or treated with anti-VCAM-1 mAb, anti-VLA-4 mAb (closed bars) or control rat IgG (open bars) and infected with 2×10⁷<i>L. donovani</i> amastigotes i.v. Mice were injected i.p. with 1 mg of antibody prior to infection and every 3 days thereafter (black bars), or starting from day 3 and every 3 days thereafter (grey bars). Parasite burdens were determined in the liver at day 14 p.i. (A) and data represent the mean±SEM in Leishman Donovan units. The number and maturity of hepatic granulomas was estimated on day 14 liver sections labelled with anti-<i>L. donovani</i> sera from VLA-4 blocked mice (B) and VCAM-1 blocked mice (C). Data represent the frequency of infected Kupffer cells (KC), immature granulomas (IG) and mature granulomas (MG) per mouse. Hepatic MNC were isolated from naïve (hatched bars) and infected mice on day 14 p.i. and enumerated (D). One representative experiment of two performed with similar outcome is shown (n = 4 mice per treatment group in each experiment). Statistical differences of p<0.01 (**) for control versus anti-VCAM-1 or anti-VLA-4 treatment are indicated.</p

VCAM-1/VLA-4 interactions are required for parasite-induced IL-12p40 mRNA accumulation by CD8⁺ DC.

<p>(A) C57BL/6 mice were treated with 1 mg anti-VCAM-1 mAb, anti-VLA-4 mAb or control rat IgG or (B) with 1 mg anti-VCAM-1 Fab fragments or control rat IgG Fab fragments the day prior to infection with 1×10⁸<i>L. donovani</i> amastigotes. CD11c⁺ DC were enriched by positive selection by MACS from the spleens of naïve mice (hatched bars), or antibody treated mice (closed bars) or control treated mice (open bars) at 5 hr p.i. mRNA was extracted from MACS-enriched DC, and accumulation of IL-12p40 mRNA was detected by real-time RT-PCR. One representative experiment of four performed with similar outcome is shown (n = 4 mice per treatment group in each experiment). All data is expressed as IL-12p40 mRNA molecules per 1000 HPRT molecules. (C–D) MACS-enriched CD11c⁺ DC from naïve C57BL/6 mice (hatched bars) or <i>L. donovani</i>-infected mice at 5 hr p.i. treated with either control rat IgG (open bars) or anti-VCAM-1 (closed bars), were sorted based on CD11c and MHC-II expression, followed by separation into CD8α-positive and CD8α-negative populations. Percentages of each gated population are indicated. (D) mRNA was extracted from purified CD8⁺ or CD8⁻ DC populations, as indicated, and accumulation of IL-12p40 mRNA was detected by real-time RT-PCR. Data represent groups of pooled cells from 4 mice, repeated 3 times. (E) Proliferation of splenic CD4⁺ T cells from naïve (hatched bars) or <i>L. donovani</i>-infected C57BL/6 mice (day 14 p.i.) that had received anti-VCAM-1 mAb, anti-VLA-4 mAb (closed bars) or control rat IgG (open bars), as indicated, in the presence of naïve irradiated splenic APC pulsed with fixed <i>L. donovani</i> amastigotes. One representative experiment of two performed with similar outcome is shown (n = 4 mice per treatment group in each experiment). Data is presented as a stimulation index (SI) calculated by dividing the proliferation of each sample in response to parasite antigen by proliferation in culture media alone. Statistical differences of p<0.01 (**) for antibody versus control treated mice are indicated.</p