Figure 1.
PPD antigen-bead pulmonary granuloma model.
(A) Schematic representation (rbead: radius of bead, rg: radius of granuloma) and (B) histological appearance of an artificial pulmonary granuloma induced in mouse 4 days after injection of PPD-coated beads [37], [38], [41] (H&E staining; magnification: ×800).
Figure 2.
Schematic representation of the multi-scale two-compartment model of PPD bead granuloma and TNF-associated reactions.
(A) Binding interactions and reactions controlling TNF/TNFR dynamics at the single-cell level, including synthesis of TNFR1, TNFR2 and mTNF, sTNF release to the extracellular space under the effect of TACE activity, reversible binding of sTNF to TNFR1 and TNFR2, sTNF degradation, internalization of free and sTNF-bound TNFR1 and TNFR2, degradation of internalized TNFR1 and TNFR2, recycling of internalized TNFR1 and TNFR2, shedding of sTNF-bound TNFR2 and release of sTNF from the shed sTNF/TNFR2 complex. (B) TNF neutralization-associated reactions, including reversible binding of drug to mTNF and sTNF, release of drug-bound TNF from the membrane to the extracellular space and drug degradation. (C) Two-compartment model of granuloma that includes a bead of radius rbead surrounded by the inner compartment populated by macrophages and DCs and the outer compartment composed of lymphocytes. Numbers in (A) and (B) represent model reactions as listed in Table 2.
Table 1.
Definition of reaction species.
Table 2.
Model reaction and their rates (vi).
Table 3.
Model parameters, definitions and values estimated from literature.
Table 4.
TNF neutralization-associated parameters, definitions, and values.
Table 5.
Model equations.
Figure 3.
Simulation results for the steady-state profile of sTNF-bound fraction of cell surface TNFR1 in a granuloma using seven different sample sets of parameter values within ranges specified in Table 3.
Arrow indicates radius of the bead (rbead). Parameter values for the particular curves shown are listed in Supplementary Table S2.
Table 6.
Significant PRCC values for model parameters and four spatially averaged steady-state outputs: (1) sTNF-bound fraction of cell surface TNFR1 in the whole granuloma, (2) sTNF-bound fraction of cell surface TNFR1 in the inner compartment, (3) sTNF-bound fraction of cell surface TNFR1 in the outer compartment, (4) sTNF concentration in the whole granuloma.
Figure 4.
Cellular fractions in PPD bead granulomas at 2 and 4 days of granuloma formation in thirty CBA/J mice quantified by multi-color flow cytometry.
Results are expressed as the percentage of each cell type in the total population of granuloma cells. Error bars represent standard deviation from the mean.
Table 7.
Average numbers of TNF receptors per cell quantified by multi-color flow cytometry for different types of granuloma-comprising immune cells isolated from 15 mice.
Figure 5.
Quantification of the rate of mTNF synthesis by each cell type.
Experimental data on the number of mTNF molecules on the surface of each cell type after addition of TAPI-1 were fitted to Equation 10 to estimate ksynth for that cell type. Displayed data represent TNF synthesis by day 4 granuloma cells for three hours in the presence of TAPI-1. Error bars indicate standard deviations. Values of R2 for curve fitting for mDCs, macrophages and pDCs are 0.97, 0.99 and 0.98, respectively.
Table 8.
Average rate of mTNF synthesis and average rate constant for TNF release quantified by multi-color flow cytometry for different types of TNF-expressing immune cells (isolated from 10 mice) isolated from day 2 and 4 granulomas.
Figure 6.
Predictions of the two-compartment model for a PPD bead granuloma.
(A) The effects of receptor binding, intracellular trafficking of TNF and cellular organization within granuloma (represented by separation) on the steady state spatial distribution of free sTNF in a granuloma. (B) The effect of separation between different cell types in a granuloma on the spatial concentration of sTNF-bound cell surface TNFR1. Parameter values for the rate of mTNF synthesis (and similarly for TNFR densities) in each compartment were computed via Equations 6 and 7, using experimental data for day 4 granulomas presented in Figure 4 and Tables 7 and 8. Other parameter values are as listed in Table 3. The qualitative aspects of these plots are similar for day 2 granulomas.
Figure 7.
Model predictions for the effect of TNF-neutralizing drugs with various properties on the availability of TNF within a granuloma.
(A) Class 1: the drug can only bind to sTNF with a binding ratio of 1∶1. (B) Class 2: the drug can bind to both mTNF and sTNF with a binding ratio of 1∶1. The star shows the location of a drug with TNF binding kinetics similar to etanercept. (C) Class 3: the drug can bind to both mTNF and sTNF with a binding ratio of 3∶1. The star shows the location of a drug with TNF binding kinetics similar to infliximab. (D) Model predictions for the effect of TNF/drug association rate constant on neutralization efficiency of drugs of different classes but identical affinities (Kd_Drug = koff_TNF/Drug/kon_TNF/Drug = 10−9 M). Model parameter values are the same as Figure 6. TNF neutralization-associated parameter values are as listed in Table 4.
Figure 8.
Spatial coordination of the TNF-induced immunological functions in a classical granuloma composed of a core of macrophages and DCs surrounded by a ring of lymphocytes.
Great availability of TNF in the core of granuloma (together with TNF-induced TNFR2 activation) can turn on the TNFR1-dependent caspase-mediated apoptotic pathway that favors antigen cross-presentation as well as elimination of the pathogen inside the granuloma. Low level of TNF availability in the mantle of granuloma is sufficient to turn on the NF-κB signaling which favors cell survival and expression of pro-inflammatory genes but not the apoptotic pathway.