Subjects and pulmonary function testing
A group of healthy subjects was studied (Table ) with 40 to 65 years of age (12 men, five women), which was divided into never smokers (NS) and asymptomatic smokers (S). Further, 18 outpatients with stable chronic obstructive bronchitis (COB) (10 men and 8 women; 45 to 72 years of age), 12 outpatients with idiopathic pulmonary fibrosis (IPF) (five men and seven women; 27 to 74 years of age), and 15 outpatients with pulmonary sarcoidosis (SAR) (five men and ten women; 34 to 69 years of age) participated in this study. Chronic bronchitis was defined as cough and sputum production occurring on most days of the month for at least three months/year during the two years prior to the study [25
]. Among COB patients, more than 50% were no longer active smokers and were therefore classified into a separate class of ex-smokers (XS). Anamnesis was carried out using a questionnaire based on ATS – recommendations [26
]. The smoking history of the participants was quantified using the cumulative cigarette consumption expressed in pack-years (PY). None of the healthy subjects had a history of respiratory or cardiovascular disease or was receiving any long-term medication. Only smokers with normal lung function data were enrolled in the study. 6 patients with COB were receiving long-term β2
-agonists and inhaled corticosteroids. 3 patients with COB received no medication at the time of the study. 4 patients with IPF and 2 patients with SAR were treated with oral steroids. The protocol was approved by the Ethical Committee of the Medical School of the Ludwig-Maximilian University (Munich, Germany), and informed consent from each subject was obtained.
Age, cumulative cigarette smoke consumption, and lung function data of all subjects of the study.
Body plethysmography and spirometry were performed using a Jäger Masterlab (Erich Jäger, Würzburg, Germany). Relative values of conventional lung parameters were calculated by normalizing to the reference values proposed by the European Community for Steel and Coal [27
]. The transfer factor of the lung for carbon monoxide (TL,CO
) was calculated as proposed by Cotes [28
]. A lung function test and the MPG measurement of the natural ferromagnetic contamination of the lungs of every subject were obtained before inhalation. MPG measurements were performed 30 min., two days, one week, one month, five months and nine months after particle inhalation.
Preparation of ferrimagnetic tracer-particles, inhalation and detection
The preparation, inhalation and detection of the magnetic particles is described in detail elsewhere [11
]. In brief spherical monodisperse ferrimagnetic iron-oxide particles (Fe3
, 2.9 μ
m aerodynamic, 1.35 μ
m geometric diameter, σg
< 1.1) were produced by a Spinning Top Aerosol Generator (STAG) [29
], concentrated by using a virtual impactor, and directly inhaled by the participants under standard conditions (250 cm3
/s flow rate, 1 L tidal volume). After inhalation, the subject lies on a bed with the lungs directly under the magnetizing coils. Magnetization is performed by discharging a capacitor battery into a 40 cm diameter copper coil. After the magnetizing current has decayed, the magnetized particles produce a weak remanent magnetic lung field (LF) of about 100 pT, which was detected by moving the subject under a superconducting magnetic field sensor (SQUID, superconducting quantum interference device). Particle twisting was performed by a weak magnetizing current in the 40 cm diameter copper coil, which was controlled for discrete time durations.
For MPG measurements about 1 mg of iron oxide particles were deposited in the lung. This amount is low compared to occupational exposure at work places, such as welders [30
]. In a previous study a bronchoalveolar lavage was performed in one subject 7 days after particle inhalation [32
]. In this study only single particles could be found in the alveolar macrophages, confirming the rotational behavior of single particles in cells.
Correction for non-rotational particles (phagocytosis)
During inhalation, the particles are deposited on the epithelial surface of the lungs. Over the following 24 hours, they are phagocytized by alveolar macrophages. Directly after inhalation, particle twisting in a weak magnetic field reveals an alignment asymmetry, which reflects a certain fraction of non-rotatable particles [17
]. After the particles are pulse magnetized, they are aligned with the magnetizing field. Then they are twisted by a reverse magnetic field into the opposite direction. When again reversing the twisting field, the particles are rotated to the initial direction of pulse magnetization. Free particles stack on the epithelium, are unable to follow this rotation and induce an asymmetry in the particle alignment, which allows estimating the fraction of non-rotatable particles. Directly after inhalation (30 min.), about 50% of the particles are not rotatable. This fraction decreases in the following hours to 5 – 10%. We suggest that the non-rotatable particles are not phagocytized by macrophages and stick to the alveolar epithelium. In vitro
studies with J774 macrophages have shown that all particles which are phagocytized by macrophages are rotatable in weak twisting fields. Microscopic investigations have shown that these particles are covered by surfactant, displacing them into the epithelial lineage [33
]. This tight contact to the epithelium hinders the particle rotation for a complete alignment. This in vivo
measurement of non-rotatable particles measures the phagocytosis process in the human lungs. All relaxation and particle twisting data are corrected by the fraction of non-rotatable particles.
Macrophage motility (relaxation)
The motion of vesicles and phagosomes happens permanently within living cells and is part of the intracellular transport system. Figure summarizes the phagosome motions, which can be followed in MPG. First, the particles are magnetized and aligned in a strong magnetic field pulse. Stochastic phagosome motions are caused by the intracellular transport mechanism and imply a decay of the remanent magnetic field of the lung (LF). This decay of the LF was called relaxation and was analyzed by the relative LF after 1 min., b1 = B(1 min.)/B0
, which characterizes the initial fast phase, and the relative LF after 5 min., b5 = B(5 min.)/B0
, being characteristic for the following slow phase (Figure ). These two relaxation parameters are independent of any model. Modeling relaxation as a rotational Brownian motion process in a Newtonian fluid reveals an exponential decay (see Appendix). Relaxation in living cells shows deviations from this model with an initially faster decay, which can result from viscoelasticity [34
]. For simplicity exponential functions were fitted to the initial and to the slow relaxation phase with the appropriate time constants.
Figure 1 Schematic views of magnetic particles within macrophages during the three stages of the investigation showing the direction and strength of the magnetic field, the orientation of the dipoles formed by the magnetized particles, and the decay of the measured (more ...)
Figure 2 Measurement of macrophage motility as decay of the magnetic lung field (relaxation) after particle alignment by pulse magnetization. b1 = B(1 min.)/B0 and b5 = B(5 min.)/B0 denote the relative decay of the initial phase (after 1 min.) and of the slow (more ...)
Macrophage motility (randomization energy)
We postulate a cellular energy Er
, which is the driving force of intracellular transport mechanisms and of relaxation. During particle twisting this energy acts against the magnetic aligning force and prevents a complete orientation of the particles, as will be achieved in primary (strong field) magnetization. The balance between magnetic twisting force and cellular randomization force determines an equilibrium alignment of the particles in the cells (Figure ). This equilibrium alignment has an analogy in paramagnetism and can be described by the so-called Langevin-function L(α
Measurement of cellular energy Er as competition between ordering of the dipoles in the external magnetizing field BM and randomization of the dipoles due to cytoskeletal motility.
where m is the magnetic dipole moment and BM is the external weak twisting field. This allows a direct measure of Er. The experiments were performed in a way that first, all particles were magnetized and aligned in a strong magnetic field, yielding the maximum lung field (Bmax). Relaxation was allowed over a period of 10 min., during which the lung field decayed below 50% of the initial value. Then a weak field of 1 mT strength was applied over 3 min. in order to realign the particles. The equilibrium lung field (Beq) is a measure of the achieved dipole alignment. Cellular energy Er was then estimated from Beq (BM) = Bmax·L(α). This method of estimating cell motility is independent of the viscoelastic environment.
Cytoskeletal integrity: mechanical properties of the cytoskeleton of macrophages
Application of a weak magnetic field mediates an external particle twisting (Figure ) and allows the investigation of mechanical properties of the cytoskeleton, like cytoplasmic rheology and mechanical integrity. Experiments for both discrete and continuous particle twisting were performed. Discrete particle twisting implies the force application for a short time period of 10 sec. This causes an angular shear of the particles and a measure of apparent viscosity. If the surrounding medium has elastic properties, we get elastic recoil which yields information about substructures of the cytoskeleton. During continuous particle rotation, the twisting field BM is applied for 3 min. until the dipole orientation reaches equilibrium.
Brownian rotational particle motion has an influence on particle twisting in a weak magnetic field, and, as was shown above, it prevents complete particle alignment. The influence is significant when the twisting force is small, as we have in small twisting fields, or when the magnetic dipole aligns with the twisting field, which is the case at continuous particle twisting during the equilibrium phase. For the estimation of viscous and elastic parameters, the influence of Brownian motion was neglected.
Continuous particle twisting
The experimental procedure of continuous particle twisting is shown in Figure . After strong field magnetization, 2 min. relaxation was allowed in order to rotate the dipoles a certain angle away from the initial orientation. Then a reverse twisting field was applied for discrete time periods (5 s, 10 s, 20 s, 30 s etc.), between these periods, the LF was detected by moving the subject under the SQUID-sensor and back to the magnetizing coil, which requires about 3 – 4 sec. The small amount of elastic recoil during this short time period was neglected. This procedure was continued until a total twisting duration of 3 min. was achieved. Because the LF is a measure of cosθ(t) (see Appendix), we can get an estimation of the mean orientation angle θ(t) of the particles. Particles suspended in a Newtonian viscosity η rotate in an external magnetic twisting field according to Newton's law:
Figure 4 a) Continuous twist of the magnetic microparticles after pulse field alignment and 2 min. of relaxation. b) Analysis of the corresponding shear rate dependence of apparent viscosity during continuous particle twist. ηm denotes the apparent viscosity (more ...)
where dθ/dt is the shear rate, σ is the applied shear stress. The viscosity η is the constant of proportionality. The complete course of particle twisting under certain boundary conditions is described in detail in the Appendix.
The curve NV in Figure shows particle twisting in a Newtonian viscosity (ηm
), and the time constant was adapted to the initial measurements. Increasing duration of stress application reveals a retarded particle rotation in the cells, which implies stiffening and non-Newtonian behavior of the cytoskeleton. The non-Newtonian apparent viscosity is characterized by the shear rate dependence according to Eq. A6 and is shown in Figure . Apparent viscosity increases with decreasing shear rate sr according to a power law: η
, where the power α
is near unity. This behavior is called pseudoplastic [37
] and is characteristic for particle twisting in alveolar macrophages and in J774 macrophages [38
]. Simulations have shown that this behavior originates in part by elastic properties [15
]. The previous analyses show that the first measure of apparent viscosity after 5 sec. stress duration (ηm
) is a reliable estimation of the viscosity of a more complex viscoelastic system.
Discrete particle twisting (viscoelastic recoil)
Continuous particle twisting experiments demonstrate non-Newtonian viscous properties, but do not allow a direct measure of cytoskeletal elasticity. A visualization of cytoskeletal elasticity was achieved by applying the twisting field for a short time period of 10 sec. According to Hoke's law the applied shear stress is proportional to the strain (rotation angle θ), and the constant of proportionality is the rigidity ν.
σ = ν·θ (3)
Energy being stored during this shear is recovered by elastic recoil of the particles. Modeling viscoelastic recoil by a Voigt body, in which a viscosity η and the rigidity modulus ν are in parallel (Appendix), a direct estimation of the elasticity modulus is possible. In a Voigt-body, elastic recoil returns the dipoles back to their orientation before stress application. Particle twisting in living macrophages does not yield complete recoil (Figure ); most of the strain remains non-recoverable. This required an extension of the Voigt-body by an additional viscous element η1. The whole system is then called a Voigt-Maxwell body. Non-recoverable strain can indicate non-reversible deformations in the cytoplasm.
Figure 5 Discrete reverse particle twist after pulse field alignment and 2 min. of relaxation. The pure viscous element (viscosity η1) describes the non-recoverable strain and the viscoelastic element (viscosity η2, elastic modulus ν2) (more ...)
Cell stiffness was estimated as the ratio between mean stress to strain after a constant twisting duration of 10 sec.:
This analysis of stiffness does not view for specific viscous or elastic properties. Therefore this parameter provides an integral description of the cytoskeletal mechanical properties.
Because of the influence of phagocytosis and macrophages activation on the estimation of macrophage motility and mechanical cytoskeletal properties only the data from one week after particle deposition till the end of the study were used to form mean values for cell motility and cytoskeletal mechanics. This also eliminates possible effects of particles being deposited in the airways. The SAS statistical software was used for data analysis. Because some parameters were not normally distributed, data reported in Table and Table were compared using a t-Test and a non-parametric Wilcoxon-test. Significance levels obtained with the Wilcoxon-test were mostly higher compared to the t-Test. The border of achieved significance given in the results holds for both tests, when not separately mentioned. Pearson's and Spearman's rank correlation analyses were performed in order to test possible relations between the parameters.
Results of the measurement of relaxation and of randomization energy, Er