Analysis of mouse dendritic cell migration in vivo upon subcutaneous and intravenous injection - PubMed (original) (raw)
Comparative Study
Analysis of mouse dendritic cell migration in vivo upon subcutaneous and intravenous injection
M B Lappin et al. Immunology. 1999 Oct.
Abstract
Dendritic cells (DC) have an increasingly important role in vaccination therapy; therefore, this study sought to determine the migratory capacity and immunogenic function of murine bone-marrow (BM)-derived DC following subcutaneous (s.c.) and intravenous (i.v.) injection in vivo. DC were enriched from BM cultures using metrizamide. Following centrifugation, the low-buoyant density cells, referred to throughout as DC, were CD11c(high), Iab(high), B7-1(high) and B7-2(high) and potently activated alloreactive T cells in mixed lymphocyte reactions (MLR). In contrast, the high-density cells expressed low levels of the above markers, comprised mostly of granulocytes based on GR1 expression, and were poor stimulators in MLR. Following s.c. injection of fluorescently labelled cells into syngeneic recipient mice, DC but not granulocytes migrated to the T-dependent areas of draining lymph nodes (LN). DC numbers in LN were quantified by flow-cytometric analysis, on 1, 2, 3, 5 and 7 days following DC transfer. Peak numbers of around 90 DC per draining LN were found at 2 days. There was very little migration of DC to non-draining LN, thymus or spleen at any of the time-points studied. In contrast, following i.v. injection, DC accumulated mainly in the spleen, liver and lungs of recipient mice but were largely absent from peripheral LN and thymus. The ability of DC to induce T-cell-mediated immune responses was examined using trinitrobenzenesulphate (TNBS)-derivatized DC (TNBS-DC) to sensitize for contact hypersensitivity responses (CHS) in naive syngeneic recipients. Following s.c. injection, as few as 105 TNBS-DC, but not TNBS-granulocytes, sensitized for CHS responses. However, the same number of TNBS-DC failed to induce CHS following i.v. injection. In summary, this study provides new and quantitative data on the organ specific migration of murine BM-derived DC following s.c. and i.v. injection. The demonstration that the route of DC administration determines the potency of CHS induction, strongly suggests that the route of immunization should be considered in the design of vaccine protocols using DC.
Figures
Figure 1
Metrizamide enriches DC from heterogeneous GM-CSF and IL-4-treated BM-cultures. Cells were harvested directly from day 6 BM-cultures (a), or were separated into high-density (b) or low-buoyant density (c) cell populations by metrizamide. The expression of Iab, B7-1, B7-2, CD11c, GR-1 and MAC-3 (filled symbols) along with the appropriate isotype control (open symbols) on each population was analysed by flow cytometry.
Figure 2
BM-DC migrate to the T-cell area of draining LN following s.c. injection into syngeneic recipients. DC and granulocytes were labelled with PKH2-GL green fluorescent dye (Sigma). Following extensive washing in RPMI and then PBS, 6×105 cells were injected s.c. into the hind footpads of syngeneic recipient mice (_n_=3). After 48 hr draining LN were removed, snap frozen and cryosectioned for immunohistochemical analysis. (a) A LN with fluorescently labelled DC, white bar=200 mm. (b) T lymphocytes were visualized using biotinylated rat-anti-mouse CD3 and Cy™3-conjugated streptavadin (red), white bar=50 mm. (c, d) After 1–7 days the popliteal LN (_n_=9) were removed. A single-cell suspension from pooled organs was prepared by mechanical dissaggregation, DC were enriched from these suspensions using metrizamide, and their fluorescence intensity (_x_-axis) and forward scatter (_y_-axis) was analysed. (c) The time-course of DC migration to draining LN following intradermal injection. (d) The migration of s.c. injected granulocytes to the draining LN.
Figure 3
DC home in a time-and dose-dependent manner to draining LN. In (a) 6×105 labelled DC were injected s.c. into the hind footpads of syngeneic recipient mice (_n_=5). After 1–7 days the popliteal, inguinal and auricular LN (_n_=9) along with spleens and thymi (_n_=3) were removed and a single-cell suspension was prepared from pooled organs by mechanical dissaggregation. DC were enriched from these suspensions using metrizamide, and the number of fluorescent cells was analysed. (a) The timecourse of DC migration, expressed as DC per million cells, to lymphoid organs following s.c. injection. (b) Labelled DC at the indicated concentration were injected s.c. into the hind footpads of syngeneic recipient mice (_n_=5 per group). After 48 hr, single cell suspensions from popliteal LN (_n_=10 per group) were prepared. DC numbers were analysed by flow cytometry and the results expressed as DC per million LNC.
Figure 4
DC accumulate in the spleen, liver and lung following i.v. injection. (a) DC were labelled with PKH2-GL and 1×106 cells were injected i.v. into two mice. After 24 hr, spleens, peripheral LN (_n_=24), livers, lungs and thymi were pooled and a single-cell suspension produced. DC numbers were analysed by flow cytometry and the results expressed as DC per organ±SD from two separate experiments. (b, c) Spleen was snap-frozen 48 hr after i.v. injection of PKH2-GL labelled DC. Cryosections were left untreated (b) or stained with PE-conjugated anti-CD3 antibody (c). Staining specificity was confirmed with a PE-conjugated isoptype control antibody (not shown).
Figure 5
TNBS derivatized BM-DC but not granulocytes induce CHS responses to naive syngeneic recipients. DC and granulocytes were incubated with 1 mm trinitrobenzenesulphate (TNBS) at 37° for 15 min. Following extensive washing with cRPMI and then PBS, cells were resuspended in PBS and various concentrations of cells were injected s.c. into the shaved abdomen of C57BL/6 mice (_n_=5). Positive and negative control mice (_n_=5) were painted on the abdomen with 100 ml of 7% Trinitrochlorobenzene (TNCB) or vehicle (4:1 acetone:olive oil), respectively. Five days later the ears of all mice were measured prior to challenge on the dorsum of both ears with 20 μl 1% TNCB in vehicle. The graph shows data for the 24 hr timepoint as the mean increase in ear thickness±standard error mean (SEM) (_n_=10).
Figure 6
DC induce CHS following s.c. but not i.v. administration. DC were derivatized with TNBS, and following extensive washing, 105 cells were injected s.c. or i.v. into naive syngeneic C57BL/6 recipient mice (_n_=5). Positive and negative control mice (_n_=5) were painted on the abdomen with 100 μl of 7% TNCB or vehicle (4:1 acetone:olive oil), respectively. CHS responses were elicited on day 5, the mean increase in ear thickness±SEM for the 24 hr time-point are shown.
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