Differential effects of serum heat treatment on chemotaxis and phagocytosis by human neutrophils

Abstract

Neutrophils, in cooperation with serum, are vital gatekeepers of a host's microbiome and frontline defenders against invading microbes. Yet because human neutrophils are not amenable to many biological techniques, the mechanisms governing their immunological functions remain poorly understood. We here combine state-of-the-art single-cell experiments with flow cytometry to examine how temperature-dependent heat treatment of serum affects human neutrophil interactions with "target" particles of the fungal model zymosan. Assessing separately both the chemotactic as well as the phagocytic neutrophil responses to zymosan, we find that serum heat treatment modulates these responses in a differential manner. Whereas serum treatment at 52°C impairs almost all chemotactic activity and reduces cell-target adhesion, neutrophils still readily engulf target particles that are maneuvered into contact with the cell surface under the same conditions. Higher serum-treatment temperatures gradually suppress phagocytosis even after enforced cell-target contact. Using fluorescent staining, we correlate the observed cell behavior with the amounts of C3b and IgG deposited on the zymosan surface in sera treated at the respective temperatures. This comparison not only affirms the critical role of complement in chemotactic and adhesive neutrophil interactions with fungal surfaces, but also unmasks an important participation of IgGs in the phagocytosis of yeast-like fungal particles. In summary, this study presents new insight into fundamental immune mechanisms, including the chemotactic recruitment of immune cells, the adhesive capacity of cell-surface receptors, the role of IgGs in fungal recognition, and the opsonin-dependent phagocytosis morphology of human neutrophils. Moreover, we show how, by fine-tuning the heat treatment of serum, one can selectively study chemotaxis or phagocytosis under otherwise identical conditions. These results not only refine our understanding of a widely used laboratory method, they also establish a basis for new applications of this method.

Figure 1. Example histograms of the fluorescence intensity of stained zymosan particles measured by flow cytometry.

(A) Results of staining with FITC-conjugated anti-C3b. (B) Results of staining with Alexa-Fluor®-488-conjugated anti-IgG. In all cases, the particles had been incubated in three different buffers, i.e., serum-free HBSS (“plain”), HBSS with 10% untreated autologous serum (37°C-ASPO), and HBSS with 10% autologous serum that had been heat-treated at 56°C (56°C-ASPO). The legend included in (A) applies to all panels.

Figure 2. Summary of the FCM assessment of the temperature-dependent deposition of C3b and IgG onto the surface of serum-opsonized zymosan.

(A) Results of staining with FITC-conjugated anti-C3b. (B) Results of staining with Alexa-Fluor®-488-conjugated anti-IgG. The first three columns depict example data obtained with sera from different, healthy donors. The rightmost column summarizes the results for a total of 3 donors. Values are block-centered means ± SD.

Figure 3. Single-cell/single-target experiment to quantify the purely chemotactic activity of non-adherent immune cells.

(A) Using dual-micropipette manipulation, a zymosan particle is stepwise brought into close proximity of an initially passive neutrophil. In this configuration, chemotaxis takes the form of a cellular pseudopod that protrudes toward the zymosan particle and responds quickly to relocation of the particle. Relative times are included in each videomicrograph. (B) Analysis of the protrusion and retraction of pseudopods during complement-mediated chemotaxis of a neutrophil. The videomicrograph depicts the cell shortly after the zymosan particle had been relocated from position (1) to (2). Also shown is the positional trace of the tip of a pseudopod, where the time of particle relocation is indicated by a color change (red → blue). Thus the blue trace follows the (mainly retracting) pseudopod after removal of the local chemoattractant source from position (1). (C) Cumulative traces obtained sequentially for three target positions. (D) Time elapsed between the placement of target particles at the given distance and the initiation of a chemotactic pseudopod local to the target. (E) Average target distance at which pseudopod formation started within 3 minutes. (F) Maximum length of chemotactic pseudopods. The experiments analyzed in (D)-(F) were performed in buffer containing 48°C-treated serum. Scale bars in (A) and (B) denote 10 µm. Values in (D)–(F) are means ± SD.

Figure 4. Single-cell/single-target experiment to quantify the time course of phagocytosis of initially quiescent immune cells.

(A) Using dual-micropipette manipulation, a zymosan particle is “handed over” to an initially passive neutrophil. Subsequent videomicrographs illustrate the ensuing phagocytosis (relative times are included). An arrow marks the small pseudopodial “pedestal” that initially “pushes” the particle outwards. In this experiment, the zymosan particle was pre-opsonized with 48°C-treated serum and then washed. The experiment buffer was supplemented with 52°C-treated serum. (B) Same type of experiment as in (A), but here the particle was opsonized in the experiment buffer containing 52°C-treated serum. (C) Quantitative comparison of typical time-dependent positional traces of opsonized zymosan particles during single-cell phagocytosis by initially passive neutrophils. Two traces each are shown for zymosan opsonized with 48°C- and 52°C-treated serum, respectively. The individual time axes were aligned for maximum overlap during the inward movement of the particles. The inset illustrates our analysis in terms of the maximum push-out distance of the target particle and of the total engulfment time. (D) Average maximum push-out distances of zymosan from measurements such as illustrated in (C) for the two types of opsonized zymosan particles. (E) Average target engulfment times for the same experiments as analyzed in (D). Scale bars in (A) and (B) denote 10 µm. Also included in (D) and (E) are the results of previous measurements (shown in a lighter color) where zymosan had been opsonized in 56°C-treated serum. Values in (D) and (E) are means ± SD.

Figure 5. FCM analysis of bulk interactions of human neutrophils with zymosan particles in differentially heat-treated serum (HTS).

(A) Plots of forward versus side scatter were used to delineate the region of FCM events that contains neutrophils. The scatter data of purified neutrophils (top panel) were included in all other panels for comparison (gray symbols). (B) Forward scatter height-versus-width density plots of the FCM events of the neutrophil gate defined in (A). These plots were used to further discriminate single-neutrophil events from coincidence events. (C) Histograms of the FITC fluorescence intensity of the events of the gate of (B) reveal varying degrees of neutrophil association with fluorescent zymosan particles. Also shown are the respective data for isolated neutrophils (obtained in the absence of zymosan; “iso-PMN”; top row), and for a mixture of neutrophils and un-opsonized zymosan (negative control; “plain”; bottom row).

Figure 6. Example measurements of the uptake of fluorescent zymosan particles by human neutrophils.

Fluorescence data such as shown in Fig. 5C were used to determine the mean fluorescence per neutrophil (this average includes all neutrophils), as well as the fraction of neutrophils that have associated with at least one zymosan particle, as a function of the serum-treatment temperature.

Figure 7. Summary of the FCM assessment of interactions between neutrophils (PMNs) and fluorescent zymosan particles in suspension as a function of serum heat treatment.

The first three columns depict example data obtained with neutrophils and autologous sera from three different, healthy donors. The rightmost column summarizes the results for a total of 6 donors. Values are block-centered means ± SD.