Quantitative analysis of the role of fiber length on phagocytosis and inflammatory response by alveolar macrophages

Abstract

Background: In the lung, macrophages attempt to engulf inhaled high aspect ratio pathogenic materials, secreting inflammatory molecules in the process. The inability of macrophages to remove these materials leads to chronic inflammation and disease. How the biophysical and biochemical mechanisms of these effects are influenced by fiber length remains undetermined. This study evaluates the role of fiber length on phagocytosis and molecular inflammatory responses to non-cytotoxic fibers, enabling development of quantitative length-based models.

Methods: Murine alveolar macrophages were exposed to short and long populations of JM-100 glass fibers, produced by successive sedimentation and repeated crushing, respectively. Interactions between fibers and macrophages were observed using time-lapse video microscopy, and quantified by flow cytometry. Inflammatory biomolecules (TNF-α, IL-1α, COX-2, PGE₂) were measured.

Results: Uptake of short fibers occurred more readily than for long, but long fibers were more potent stimulators of inflammatory molecules. Stimulation resulted in dose-dependent secretion of inflammatory biomolecules but no cytotoxicity or strong ROS production. Linear cytokine dose-response curves evaluated with length-dependent potency models, using measured fiber length distributions, resulted in identification of critical fiber lengths that cause frustrated phagocytosis and increased inflammatory biomolecule production.

Conclusion: Short fibers played a minor role in the inflammatory response compared to long fibers. The critical lengths at which frustrated phagocytosis occurs can be quantified by fitting dose-response curves to fiber distribution data.

General significance: The single physical parameter of length can be used to directly assess the contributions of length against other physicochemical fiber properties to disease endpoints.

Keywords: Frustrated phagocytosis; Glass fibers; Length; Macrophage; TNF-α: Tumor necrosis factor-α.

Figure 1. Fiber length distribution

Representative histograms of the length distributions of (a) short and (b) long fibers. Both populations exhibit a log-normal distribution for fiber lengths (inset).

Figure 2. Time-lapse video microscopy frames of macrophage-fiber binding events

(A) A short fiber being internalized by a macrophage. (B) A macrophage attaching to and pulling a long fiber toward itself without internalization. Scale bar: 20 μm, Time: 0–3 hrs.

Figure 3. Fiber-cell interactions with increasing short fiber dosage

Fiber-cell interactions quantified by flow cytometry reveal a dose-dependent increase in all interactions for short fibers. The relative percentage of cells with internalized interactions compared to total interactions was relatively unchanged for all fiber doses, with 36.4%, 35.7%, and 34.9% for short fiber doses 5, 10, and 20 respectively. A total of 10,000 cells were counted including cells with no associated fibers.

Figure 4. Cytotoxicity of macrophages exposed to short and long fibers

The percentage cytotoxicity was measured as the quantity of LDH released from fiber-damaged cells relative to a lysed cell control. Normal cell turnover corresponds to 0 fibers /cell and is noted by a dashed line (---). On average fiber cytotoxicity was not significant for both short and long fiber populations as compared to the media blank with the exception of the maximum long fiber dose, 15 long fibers/cell. *p<0.05

Figure 5. TNF-α dose-response curves

TNF-α secretion showed a linear dose response for short and long glass fibers. Data shown was collected for three (3) independent experiments.

Figure 6. IL-1α dose-response curves

IL-1α secretion showed a linear dose response for short and long fibers. Data shown was collected from three (3) independent experiments.

Figure 7. COX-2 Enzyme Production

COX-2 production decreased with increasing fiber dose -short fiber stimulation resulted in significantly high expression of COX-2 while long fibers were generally comparable to no fiber stimulation.

Figure 8. Prostaglandin E2, PGE₂, production 24 hours post-incubation

Short fiber response was independent of fiber concentration. Long fibers showed weak dose dependence in comparison to 0 fibers/cell control.