1. Introduction

In traditional Chinese herbal medicines, Reishi which belongs to a group of medical fungus has been used for promoting good health, vitality, and longevity [1]. The active constituents responsible for these effects have been qualitatively described and the structure has been partially determined [2]. Extract of Reishi polysaccharides (EORP) has been demonstrated to exert immunity modulating activities by stimulating the expression of inflammatory cytokines [3]. In western medical sciences, immunology has been extensively studied and the important role macrophages play in innate immunity has been identified in recent years. The EORP-mediated signaling pathways involved in the pro-IL-1/IL-1 regulation, have been observed in murine macrophages; Toll-like receptor 4 (TLR4) which mediates the consequent immunity modulating events associated with IL-1 gene expression is a putative receptor of EORP [3]. TLR4 and another receptor in macrophages, known as CD14, recognize and interact with lipopolysaccharide (LPS) which is one of the cell wall components of Gram-negative bacteria [4]. Such a process plays an important role in our innate immune system. In this paper, we report our study of the influence of EORP on macrophages by several methods, including flow cytometry, confocal microscopy, and optical tweezers. The experimental results provide a logical cell-biological explanation for some medical effects of Reishi.

Optical tweezers were first reported by Ashkin et al. in 1986 [5] almost 16 years after the first report on optical acceleration and trapping of micro-particle in a counter-propagating dual-beam configuration by Ashkin et al. in 1970 [6]. Optical tweezers with near infrared (NIR) laser beam (e.g., at wavelength=1.06 µm) were soon demonstrated for non-invasive trapping and manipulation of a single living cell [7]. Optical trapping and manipulation has since proven to be a useful tool in many research disciplines [8, 9]. Recently, optical trapping have been applied to biological studies to monitor molecular and cellular interactions. For example, optical trapping has been used to study the bio-molecular interactions such as those between protein-coated micro-particles and cellular plasma membranes for potential biological applications [10]. In this study, we demonstrated that EORP enhances cell surface expression of TLR4 and CD14 on macrophages as well as increases LPS binding and uptake by macrophages, and used optical tweezers to study the influence of EORP on macrophages by monitoring the interaction force and time constant of LPS binding to the plasma membrane of macrophages.

2. Materials and methods

2.1 Macrophage preparation

Murine macrophage J774A.1 cells (J774A.1 cells) were obtained from American Type Culture Collection (Manassas, VA). Cell cultures were propagated in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT) and 2 mM L-glutamine (Invitrogen Life Technologies, Carlsbad, CA) and cultured in a 37°C, 5 % CO₂ incubator. Cells were stripped on a cover glass (25 mm×25 mm) in the culture wells (60 mm diameter) for 24 hours incubation.

2.2 LPS coating on polystyrene particles

We took 2 µl plain polystyrene particles (1.5 µm diameter, concentration 10%, PS04N/6066, Bangs Laboratories, Inc., Fishers, IN) and washed (13,000 rpm, 4°C, 5 minutes) the particles twice with 1 ml bicarbonate-buffer composed of 0.05 M Na₂CO₃ and 0.05 M NaHCO3, adjusted with Na₂CO₃ to pH 9.6. We added 500 µg of LPS (50 µg/ml, from Escherichia coli 0111:B4, Sigma-Aldrich) or bovine serum albumin (BSA) with 10 ml bicarbonate-buffer and slowly rotated the content for two hours. After washing twice with 1ml bicarbonate-buffer, we added phosphate buffered saline (PBS) with 5 % BSA to reach 1ml solution and slowly rotated the content for one hour. After washing twice with 5 % BSA/PBS, we added 5 % BSA/PBS with 0.01 % NaN₃ to reach 1 ml and stored at 4°C. The protocol for the coating procedure described above was provided by Dr. Holger Kress (European Molecular Biology Laboratory, Heidelberg, Germany) [10]. In order to verify the successful coating of LPS or BSA on polystyrene particles, we coated ~10 nm gold layer on polystyrene particle for scanning electron microscope (SEM; Hitachi S-2700) imaging. This was achieved by the standard protocol (30 mA of current for 80 sec.) prescribed by the vacuum evaporator (JEE-400) for examination via SEM. Note that gold-coating on polystyrene particles was solely for SEM imaging [Fig. 1(a)]; since gold-coating was not applied to the polystyrene particles used in the trapping experiments, it had no effect at all on our experimental results. Further, to check the response of J774A.1 cells to polystyrene particles, we added polystyrene particles (either uncoated, or BSA-coated, or LPS-coated particles) into the cultured macrophage. After 6 hours, pro-IL-1 expression in cell extract was measured by Western blot [11]. We detected pro-IL-1 production in cases where the J774A.1 cells were treated with either LPS or LPScoated polystyrene particles, but not in the control case (where the J774A.1 cells were kept in a control environment) and in cases where the J774A.1 cells were treated with either BSA-coated particles or uncoated-particles [Fig. 1(b)].

 

Fig. 1. (a). SEM images (20,000X) of uncoated polystyrene particles, BSA-coated polystyrene particles and LPS-coated polystyrene particles. (b) Result of Western blot indicating the presence of pro-IL-1 where the J774A.1 cells were treated with LPS, LPS-coated, BSA-coated and uncoated polystyrene particles.

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2.3 Flow cytometric analysis

For flow cytometric analysis of the effect of EORP on macrophages in (i) cell surface expression of TLR4 and CD14, (ii) binding of LPS to cell surface, and (iii) LPS uptake by macrophages, J774A.1 cells were incubated in standard cell culture medium without EORP (for control) and with EORP (25 µg/ml) for 24 hours. To analyze the cell surface expression of TLR4 and CD14, cells were fixed and stained with PE-conjugated anti-TLR4 antibody or PE-conjugated anti-CD14 antibody on ice for 30 minutes, respectively. After washing, cells were subjected to flow cytometric analysis on FACSCalibur using CellQuest software of Becton Dickinson Inc. (San Jose, CA). To examine the binding of LPS to cell surface, J774A.1 cells were washed and incubated with FITC-LPS for 30 minutes at 4°C and were subsequently subjected to flow cytometric analysis after re-washing. To measure the LPS uptake, J774A.1 cells were washed and incubated with FITC-LPS for 1 hour at 37°C. After incubation, cells were washed twice with PBS and further treated with proteinase K (250 µg/ml) for 30 minutes at room temperature to remove cell surface proteins/receptors and surface-bound LPS [12]. Hence, the remaining LPS were most likely intracellular; the FITC-LPS in J774A.1 cells were measured by flow cytometric analysis.

2.4 Confocal microscopy analysis

In addition to the flow cytometric analysis outlined above, we also used confocal microscope to re-examine the influence of EORP on macrophages in surface expression of TLR4 and CD14, and in LPS uptake. For confocal microscopy J774A.1 cells, incubated in medium with EORP (25 µg/ml) and without EORP for 24 hours as is described in the previous section, were fixed with 2 % paraformaldehyde at room temperature for 30 minutes. After fixation, cells were blocked with 2 % BSA at room temperature for 30 minutes, followed by staining with PE-conjugated anti-TLR4 antibody or PE-conjugated anti-CD14 antibody at room temperature for 2 hours. Cells were then washed and examined with a confocal microscope (TLS SP2, Leica Lasertechnik, Heidelberg, Germany). Likewise, for LPS uptake experiments, cells were washed and incubated with TLR4 blocking antibody (10 µg/ml), CD14-blocking antibody (10 µg/ml), control antibody (10 µg/ml), cytochalasin D (10 µM) or colchicines (30 µM) for 30 minutes, followed by incubation with FITC-LPS for 1 hour at 37°C. Cells were then washed and examined with confocal microscope. Our experimental results, obtained by flow cytometry and confocal microscopy described in this and the previous sections are given in Section 3.

2.5 Optical tweezers setup

A schematic diagram of the optical tweezers setup is illustrated in Fig. 2. A linearly polarized laser beam (λ=1064 nm, 300 mW, Nd: YVO₄ cw laser, LeadLight Technology, Inc.) for optical trapping passed through a half-wave plate (HW) and a polarizer (PZ) so that the trapping laser power could be adjusted by rotating the half-wave plate. A beam expander (BE) was used to expand and collimate the beam such that the beam diameter (~1 cm) slightly overfilled the back aperture of the microscope objective. A telescope (telescope1 consisting of two lenses, each with a focal length of 150 mm) was used to change the trapping beam pathways to manipulate the trapped particle by moving the first lens (in telescope 1) amounted on a motorized translational stage (850F/PMC200-P2, Newport) in a direction perpendicular to the beam axis. The telescopic imaging arrangement transforms the transverse displacement of the first lens into a lateral shift in the focal spot of the trapping beam without any beam walk-off at the entrance pupil of the objective lens. Alternatively, the lens can also be oscillated via a piezo-electric (PZT, NS511-N, Nano Control) stage driven by a function generator (a built-in function of the Stanford Research SR-830 lock-in amplifier), and a PZT amplifier (PH301, Nano Control). A second laser (λ=632.8 nm, 10 mW, He-Ne cw laser, Uniphase) provided a tracking beam to track the motion of the trapped particle. A spatial filter-beam expander (SF/BE) unit expanded and collimated the tracking beam to a beam diameter ~6 mm. A second telescope (telescope 2) was used to adjust the position of the focal point of the tracking beam in the sample chamber with respect to that of the trapping beam. The two laser beams were combined by a dichroic mirror (DM1), which is highly reflective at λ=632.8 nm and highly transmissive at λ=1064 nm, and injected into the back aperture of a water immersion microscope objective (NA=1.0, 100X, working distance=0.97 mm, Zeiss). The tracking beam diffracted off (and scattered by) the trapped particle was collected by a condenser (NA=0.65, 40X, working distance=0.6 mm, Newport) and projected onto a quadrant photodiode (QPD, S7479, Hamamatsu) to track the motion of the particle on the transverse plane. A notch filter was used to block the trapping beam (λ=1064 nm) from entering the QPD. The electrical signals from the QPD were recorded by a data acquisition system (DAQ). For fluorescence excitation, a mercury lamp (Hg Lamp), along with a band pass filter (460–490 nm, BP460–490, Olympus), was used to inject a blue light into the water immersion microscope objective through the second and third dichroic mirror reflection (DM2/DM3, DM570 from U-MWG2/DM500 from U-MWB2, Olympus). The wide-field images of the trapped particle were captured by a CCD camera (WAT-120N, Watec) for optical alignment of the trap and for image observation and analysis.

 

Fig. 2. A schematic diagram of the experimental setup. HW: half-wave plate; PZ: polarizer; BE: beam expander; SF/BE: spatial filter-beam expander; DM: dichroic mirror; QPD: quadrant photodiode; DAQ: data acquisition system; Hg Lamp: mercury lamp.

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3. Experimental procedures, results, and discussion

3.1 Effect of EORP on macrophages phagocytosis of E. coli and binding of LPS

The influence of pre-treatment of J774A.1 cells with EORP on the interaction of LPS and CD14 on macrophage membrane was investigated as follow. Macrophage can be activated by phagocytosis of bacteria or of bacterial products such as LPS. We fed J774A.1 cells with fluorescence conjugated-E. coli and compared active intake of E. coli by J774A.1 cells pre-treated with EORP and by those without EORP treatment. We clearly observed that pre-treatment of J774A.1 cells with EORP did enhance its phagocytosis activity. J774A.1 cells without EORP treatment were much more lethargic compared with those pre-treated with EORP (video not shown). With flow cytometry, we observed a relatively fast intake of E. coli by EORP pre-treated J774A.1 cells in the initial stage (~5 minutes after E. coli feeding) compared with the cells without EORP treatment; however, the difference became insignificant at a later stage (~1 hour after E. coli feeding) [Fig. 3].

 

Fig. 3. The effect of EORP on the phagocytosis of Escherichia coli by murine J774A.1 macrophages measured by flow cytometry; (a) short term (5 minute after feeding); (b) long term (1 hour after feeding). The curves labeled “no E. coli” represent the results when the cells were not fed with E. coli. The rest of the curves were obtained (a) 5 minute and (b) 1 hour after the cells (with vs. without EORP treatment) were fed with E. coli. These data are representative of three independent experiments.

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With confocal microscopy, we observed that EORP did enhance the surface expression of CD14 on J774A.1 cells in comparison with the control cells [Fig. 4(a)]. In addition, EORP also enhanced the surface expression of TLR4 on J774A.1 cells in comparison with the control group [Fig. 4(b)]. Moreover, we also used flow cytometry analysis to double-check these results and further re-confirmed that EORP did increase the surface expressions of CD14 from approximately 5 % to 20 % and TLR4 from approximately 5 % to 32 % on J774A.1 cells as shown in Fig. 4(c).

 

Fig. 4. The enhancement of surface expression of CD14 and TLR4 on the cellular membrane of macrophage after EORP treatment; (a) CD14 expression analyzed by confocal microscopy; (b) TLR4 expression analyzed by confocal microscopy; (c) CD14 and TLR4 expression analyzed by flow cytometry. The values represent the percentage of cells with fluorescence intensity (of CD14 or TLR4 expression) higher than the reference fluorescence intensity (indicated by the dotted line). These data are representative of three independent experiments.

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For experiments designed to examine the uptake of LPS by J774A.1 cells, macrophages, with and without EORP treatment, were separately fixed and incubated with either CD14 blocking antibody (10 µg/ml), or TLR4 blocking antibody (10 µg/ml), or cytochalasin D (10 µM, to disrupt actin filaments) or colchicines (30 µM, to disrupt microtubule) for 30 minutes, followed by incubation of LPS-FITC for 30 minutes at 37°C. After washing, cells were examined with a Leica confocal microscope. We verified that the surface binding of LPS by J774A.1 cells were substantially greater for J774A.1 cells pre-treated with EORP in comparison with the control case [Fig. 5(a)]. To further analyse the role of CD14 and TLR4 in LPS binding on J774A.1 cells, the cells pre-treated with EOPR were incubated with CD14 blocking antibody or with TLR4 blocking antibody prior to LPS-FITC stimulation. We found that CD14 blocking antibody, but not TLR4 blocking antibody, significantly reduced the binding of LPS to J774A.1 cells surface, indicating that CD14 plays a more important role than TLR4 in LPS binding to J774A.1 cells [Fig. 5(b)]. In order to distinguish whether EORP-mediated J774A.1 cells up-regulation of LPS uptake was via a passive entry process or a CD14-dependent classical form of phagocytosis, we treated the cells with drugs known to inhibit cytoskeleton polymerization, such as cytochalasin D and colchicine, and examined the cells with a fluorescence confocal microscope. Micrographs taken from a confocal microscope indicated that the presence of cytochalasin D and colchicine significantly blocked the internalization of LPS-FITC by J774A.1 cells pre-treated with EORP [Fig. 5(c)] in comparison with the corresponding results for the J774A.1 cells pre-treated with EORP but not treated with cytochalasin D/colchicine.

 

Fig. 5. Phase contrast and fluorescence Images of LPS-FITC binding on J774A.1 cells analyzed by confocal microscopy; (a) cells pre-treated with EORP; (b) cells pre-treated with EORP followed by incubation with anti-CD14 or anti-TLR4 blocking antibody; (c) cells pre-treated with EORP followed by incubation with cytochalasin D or colchicine. These data are representative of three independent experiments.

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To better characterize the influence of EORP on J774A.1 cells, we measured via optical tweezers the interaction of LPS, purified from E. coli and coated on polystyrene particles, with CD14 and TLR4 on J774A.1 cells membrane, as is described in the following sections.

3.2 Interaction of LPS and macrophages measured via Brownian motion

We used optical tweezers to quantify the interaction of LPS with macrophage as follow. A LPS-coated polystyrene particle (d=1.5 µm) was trapped by optical tweezers and its Brownian motion was tracked via a QPD. The trapping optical power was initially adjusted to approximately 15 mW. The Brownian motion of the trapped particle, deduced from the calibrated QPD output signal, was found to be around ± 50 nm. A live macrophage, seated on a cover glass, was moved 50 nm per step by a PZT-driven sample stage towards the trapped particle until the trapped particle was pushed away by about 50nm from the original equilibrium position as was deduced from the pre-calibrated QPD signal. The sample stage, along with the macrophage, was then moved backwards by about 100 nm such that the distance between the trapping center and the confronting edge of the cell was around 50 nm and the trapping laser power was immediately reduced to 1 mW to allow a larger extent of the Brownian motion of the trapped particle which was measured to be around ±150 nm. The QPD output signal (data sampling rate=20,000 per second; integration time for fluctuation averaging=0.1 second) was recorded to track the particle motion and to monitor in real-time the dynamics of the binding of the particle with a macrophage. The interaction between a LPS-coated particle and a J774A.1 cell not treated with EORP is shown in Fig. 6(a); that between a LPS coated particle and a macrophage pre-treated with EORP for 24 hours is shown in Fig. 6(b). In each case, an interaction time constant τ, which is the inverse of the interaction rate, was deduced by fitting the experimental data with the mathematical expression A[1-e-^(t/τ)] where “τ” is the time constant characterizing the interaction of the particle with the macrophage, and “A” represents the initial distance between the particle and the cell. By “the steady state”, we meant the condition when the relative position of the particle remained essentially unchanged for a period on the order of 30sec. as shown in Fig. 6. For the specific example depicted in Fig. 6, the time constant “τ” characterizing the interaction of LPS coated particle with macrophage was measured to be around 4.8 second and the steady state position “A” was measured to be around 146 nm when the J774A.1 cell was not pre-treated with EOPR compared with τ=1.2 seconds and A=145 nm in cases when the J774A.1 cell was pre-treated with EOPR. In this example, the time constant “τ” characterizing the interaction of LPS coated particle with macrophage thus decreased from 4.8 seconds to 1.2 seconds when the macrophage was treated with EORP for 24 hours. The experiments described above with the control cells (without EORP treatment) and also with the EORP-pretreated cells were both repeated three times; the interaction time constant “τ” was measured to be 5.8±1.2 sec. for cells without EORP treatment and 1.05±0.18 sec. for those pre-treated with EORP.

 

Fig. 6. The relative position of the particle vs. time for the interaction of a LPS-coated particle with a J774A.1 cell; (a) for cell not treated with EORP; (b) for cell pre-treated with EORP for 24 hours. The exponential time constant “τ” associated with the binding rate, deduced from these data, was approximately τ=4.8 seconds in case (a) and τ=1.2 seconds in case (b).

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Although we could measure the dynamics of the interaction of a LPS coated polystyrene particle with the plasma membrane of a J774A.1 cell, the reproducibility of the experimental results from one measurement to another was extremely poor. In many occasions (for almost 90 % of the time), we did not observe any binding between the macrophage and the LPS-coated particle. We speculated that inhomogeneous distribution of receptors on the cell membrane could be one of the main factors. For example, inhomogeneous expression of TLR4 on macrophages was observed by treating the cell with anti-TLR4-FITC as shown in Fig. 7(a) and the binding of LPS-coated particles, preferentially in specific areas on the cell membrane is shown in Fig. 7(b). Fluorescence labeling of receptors will thus help to identify the specific locations on the cell membrane where the interactions (binding or uptake) are more probable. Additional experimental data showing variation in particle-cell interaction at different location on the membrane of the same cell are given in the next section.

 

Fig. 7. Inhomogeneous distribution of (a) TLR4, and (b) LPS-coated particles binding on J774A.1 cells membrane.

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3.3 Interaction of LPS and macrophages measured via oscillatory optical tweezers

We used oscillatory optical tweezers [13–16] to study the force constant of the binding of LPS on the plasma membrane of a J774A.1 cell. A LPS coated 1.5 µm polystyrene particle adhered to the cellular membrane was optically trapped (with trapping optical power=13 mW) and forced to oscillate along the x-axis with an oscillation amplitude of about ±20 nm, and an oscillation frequency of 10 Hz by oscillating the focal spot of the trapping laser beam. We changed the trapping beam pathways to manipulate and oscillate the trapped particle by moving the first lens (in telescope1) amounted on a piezo-electric (PZT, NS511-N, Nano Control) stage driven by a sinusoidal voltage (with V_rms=2.5 v) from a function generator (a built-in function of the Stanford Research SR-830 lock-in amplifier), followed by a PZT amplifier (PH301, Nano Control). The integration time for the lock-in detection was 1 second. Since the oscillation frequency was fairly low (i.e., 10 Hz), and the phase-sensitive detection from the lock-in amplifier picked up essentially the fundamental harmonic of the oscillation and effectively filtered out the higher harmonics, the frequency response of the PZT and other nonlinear effects are expected to be insignificant.

We analyzed the amplitude (D) and the phase shift (δ) of the oscillating particle to determine the force constant of the binding of LPS on the plasma membrane of a macrophage via the following equation. [13–16]:

where the amplitude of the oscillatory trapping beam A=20 nm, k(t) is the time dependent force constant of the binding interaction, k _OT is the transverse force constant of optical tweezers (in the absence of the cell) which was measured to be 47.5 pN/µm by analyzing the Boltzmann distribution of the particle in a parabolic potential well [16,17] when the optical trapping power was 13 mW. The corresponding transverse trapping efficiency is estimated to be ~0.1. The time dependence of the force constant of the interaction k(t) was determined from the time-dependence of the amplitude D(t) and the phase δ(t) of the oscillatory particle according to Eq. (1). Although Eq. (1) was derived for the steady state, it represented an approximate solution to the time-dependent solution in the adiabatic approximation when the rate of change of the elasticity is much slower than both the oscillation period (0.1 second in our case) and the integration time of the lock-in amplifier (1 sec. in our case). The time resolution for our measurement of k(t) by oscillatory tweezers was thus ~1 second The oscillation amplitude of the particle “D” was expressed in volt which was the output voltage (proportional the oscillation amplitude) provided by the lock-in amplifier. Since only the relative amplitude (A/D) was required to calculate the elastic constant k(t) via Eq. (1), we did not convert the amplitude (from volt) into absolute length unit in nm. However, the conversion factor was determined to be approximately 350 nm per volt. The relative phase shift “δ” of the oscillating particle with respect to that of the trapping beam was provided directly by the lock-in amplifier which compared the fundamental frequency of the signal from the QPD (which tracked the position of the trapped particle) against the PZT-driving sinusoidal voltage; as mentioned earlier, the latter was generated by a function generator, a build-in function of the lock-in amplifier.

Throughout these experiments, however, we often observed that the trapped particle was pulled off and escaped from the optical trap probably due to a combination of cell movement and phagocytosis. In order to avoid the cells phagocytosis, we treated a J774A.1 cell with EORP for 24 hours and then treated with drug cytochalasin D (known to inhibit cytoskeleton polymerizations) for 30 minutes to block the cell phagocytosis and repeated the experiment described above. The force constant of the binding interaction, defined by Eq. (1), increased as the particle was gradually pulled away from the trap center, even in the absence of phagocytosis, probably due to cell movement. As an example, experimental data showing the force constant increasing from approximately 190 pN/µm to 600 pN/µm in about 60 seconds, as the particle was gradually pulled away from the trap center, is depicted in Fig. 8(a). By repositioning the sample stage every 10 seconds via a PZT-driven stage and bringing the particle back to the trapping center of the oscillatory optical tweezers, the force constant of the binding interaction was measured to be on the order of 200 pN/µm as shown in Fig. 8(b). In Fig. 8(a), the total displacement of the particle in 60 sec. is around 0.05µm which is well within the linear range (of ~±0.2µm) of the QPD. After repeating the experiments 7 times, we decided to avoid the complexity due to the cell movement by treating the cell with paraformaldhyde as is described in the next paragraph.

 

Fig. 8. The force constant k(t) of the binding interactions of a LPS-coated particle on the plasma membrane of a J774A.1 cell pre-treated with EORP for 24 hours and then treated with cytochalasin D for 30 minutes measured as a function of time. The force constant k(t) was deduced from the amplitude “D” and the phase “δ” of the particle oscillation as is prescribed by Eq. (1); (a) The force constant increased from approximately 190 pN/µm to 600 pN/µm in about 60 seconds; (b) The force constant was measured to be approximately 200 pN/µm when the chamber was re-positioned every 10 seconds to track the particle and to keep the particle approximately at the trapping center.

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In order to avoid both phagocytosis and cell movement, we used paraformaldehyde to fix the cells and repeated the experiment; the experimental results are shown in Fig. 9. The force constant of the binding interaction was measured to be 61.5±8.8 pN/µm (Fig. 9) when the trapped and oscillated LPS-coated particle was attached to a control J774A.1 cell, which was not pre-treated with EORP. Comparatively, the force constant of the binding interaction was 226.9±67.1 pN/µm (Fig. 9) for identical experiments with J774A.1 cells pre-treated with EORP for 24 hours. We have thus demonstrated that, pre-treatment of J774A.1 cells with EORP for 24 hours, did significantly enhance the force constant of specific receptor (CD14) mediated binding of LPS-coated particles on the cellular membrane of J774A.1 cells.

 

Fig. 9. The force constant of the binding interaction of a LPS-coated particle on the plasma membrane of a J774A.1 cell pre-treated with paraformaldehyde measured by optical forced oscillation; the left bar for (control) J774A.1 cell without EORP treatment, and the right bar for J774A.1 cell pre-treated with EORP for 24 hours. The data in the control case on the left represent an average over 3 repeated experiments while the data associated with the EORPpretreated cells represent an average over 8 experiments.

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To study the variation in the force constant of the particle-cell interaction at different location on the membrane of the same cell, we selected a cell with a few particles adhered at different location on the cell membrane and measured the interaction force constant for each particle with oscillatory optical tweezers. In this experiment, the cell was treated with paraformaldehyde (to avoid cell movement) but not with cytochalsin D. Although the cell was not alive, the binding of the membrane receptors with the LPS-coated particle could still be observed. In general, we observed that more particles were adhered to the cells pre-treated with EORP in comparison with those not treated with EORP. Besides, the force constant varied widely from particle to particle adhered at different location of the same cell. As an example, force constant varying from 30 pN/µm to 200 pN/µm for 5 particles adhered to an EORP-treated cell is shown in Fig. 10(a); likewise, force constant varying from 30 pN/µm to 60 pN/µm for 2 particles in an untreated control cell is shown in Fig. 10(b). Experiments to correlate the force constant with the local concentration of receptors (CD14 and TLR4) on cellular membrane are in progress.

 

Fig. 10. LPS-coated particles binding at different location on the membrane of (a) an EORP-pretreated macrophage; (b) an untreated macrophage. The force constant of the binding interaction of a few selected particles measured by oscillatory optical tweezers are also shown in the figure.

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4. Summary and conclusion

We used confocal microscopy, flow cytometry, and optical tweezers to obtain complementary and consistent results on the influence of extract of Reishi polysaccharides (EORP) on LPS binding and intake by macrophages. The results obtained by confocal microscopy and flow cytometry clearly indicated enhanced surface expression of CD14 and TLR4 receptors on the cell membrane as well as faster intake of LPS by macrophages when the cells were pre-treated with EORP for 24 hours. With stationary optical tweezers (λ=1064 nm Gaussian beam focused by a water-immersed objective lens with N.A.=1.0), we trapped the LPS-coated polystyrene particle (diameter=1.5 µm) and measured its interaction with J774A.1 cells with and without EORP treatment. By tracking the Brownian motion of an optically-trapped LPS-coated particle interacting with a J774A.1 cell, the time constant characterizing the binding of the particle with a J774A.1 cell was determined to approximately 1.05±0.18 sec. when the cell was pre-treated with EOPR compared with 5.8±1.2 sec. in cases when the cell was not treated with EOPR. In subsequent investigations, we also measured, via optical forced oscillation of LPS-coated particles, the force constant of their binding interactions with J774A.1 cells, pre-treated with paraformaldehyde to void cell movement, and also with cytochalasin D to block phagocytosis; the elastic constant was measured to be 61.5±8.8 pN/µm when the cell was not treated with EOPR compared with 226.9±67.1 pN/µm in EORP pre-treated cell. We have thus demonstrated that EORP not only enhanced J774A.1 cells surface expression of CD14/TLR4 as well as LPS binding, but also reduced the adhesion time constant and increased the force constant of the binding interaction with a LPS-coated particle. In our experiments with optical trapping of polystyrene bead, the spatial resolution in probing different part of the cellular membrane is ~1 to 2 micron (limited by the bead diameter as shown in Fig. 10) and the temporal resolution is ~0.1 millisecond for stationary tweezers and ~1 second for oscillatory tweezers; the latter is limited by the oscillation frequency of the bead with sufficient amplitude for adequate signal-to-noise ratio in the measurement.