Adoptively transferred dendritic cells restore primary cell-mediated inflammatory competence to acutely malnourished weanling mice

Abstract

Immune depression associated with prepubescent malnutrition underlies a staggering burden of infection-related morbidity. This investigation centered on dendritic cells as potentially decisive in this phenomenon. C57BL/6J mice, initially 19 days old, had free access for 14 days to a complete diet or to a low-protein formulation that induced wasting deficits of protein and energy. Mice were sensitized by i.p. injection of sheep red blood cells on day 9, at which time one-half of the animals in each dietary group received a simultaneous injection of 10(6) syngeneic dendritic cells (JAWS II). All mice were challenged with the immunizing antigen in the right hind footpad on day 13, and the 24-hour delayed hypersensitivity response was assessed as percentage increase in footpad thickness. The low-protein diet reduced the inflammatory immune response, but JAWS cells, which exhibited immature phenotypic and functional characteristics, increased the response of both the malnourished group and the controls. By contrast, i.p. injection of 10(6) syngeneic T cells did not influence the inflammatory immune response of mice subjected to the low-protein protocol. Antigen-presenting cell numbers limited primary inflammatory cell-mediated competence in this model of wasting malnutrition, an outcome that challenges the prevailing multifactorial model of malnutrition-associated immune depression. Thus, a new dendritic cell-centered perspective emerges regarding the cellular mechanism underlying immune depression in acute pediatric protein and energy deficit.

Figure 1

Delayed hypersensitivity response to SRBCs. C57BL/6J mice, initially 19 days old, were given free access for 14 days to a complete purified diet (groups designated “C”) or to an isocaloric low-protein diet (LP groups). Mice were sensitized with SRBCs on day 9 of the experimental period and challenged by injection of the same antigen into the footpad on day 13. The immune response was assessed 24 hours after challenge by determining the increase in thickness of the challenged footpad. Groups C and LP each included 30 mice, and within each dietary group, 15 mice received 10⁶ syngeneic dendritic cells (JAWS II) by i.p. injection together with the sensitizing dose of SRBCs. Data were subjected to two-way analysis of variance with the main effects being diet and receipt of dendritic cells. Bars represent antilogs of means from log-transformed data, and SDs are shown. Error mean square = 0.993; P = 0.001 (diet), 0.001 (receipt of dendritic cells), and 0.004 (interaction). Statistical probabilities are shown for the three predetermined comparisons permitted by least squares means analysis.

Figure 2

Representative flow cytometer histograms of JAWS II dendritic cells cultured under standard conditions. Histograms from stained suspensions are shown in gray outline, and isotype control histograms are shown in black. The percentage of viable cells judged positive is recorded in each surface marker histogram. A: Forward (FSC)- and side (SSC)-angle light scatter. Box R1 identifies viable single cells and vertical dotted line shows their mean forward scatter. Phycoerythrin-conjugated anti-mouse B: I-A^{p,k,b,q,r,s,j} (7-16.17, mouse IgG2a), C: CD80 (RMMP-1, rat IgG2a), and D: CD86 (RMMP-2, rat IgG2a).

Figure 3

Concentrations of IL-10 and IL-12p70 generated by JAWS II dendritic cells cultured for 5 days in Alpha Essential Medium containing 10% fetal bovine serum and 5 ng/ml murine granulocyte-macrophage colony-stimulating factor with (+) or without (−) 10 μg/ml E. coli lipopolysaccharide (LPS) for the final 48 hours of culture. Bars represent means and SDs are shown (n = 5 and 4 per group for IL-10 and IL-12p70, respectively). Statistical probabilities are outcomes of two-tailed Student’s t-test. Horizontal dashed lines depict detection limits of each assay estimated as described previously.

Figure 4

Representative flow cytometer histogram illustrating the purity of the syngeneic CD3⁺ cells adoptively transferred into weanling C57BL/6J recipients subjected to acute protein and energy deficit and immunized to elicit an anti-SRBC delayed hypersensitivity response. Nylon wool enrichment was quantified using single-cell suspensions stained with phycoerythrin-conjugated anti-mouse CD3 (145-2C11, hamster IgG; gray outline). Isotype controls (black) were created with phycoerythrin-conjugated streptavidin after exposure of cells to biotin-conjugated hamster IgG.