Peculiarities of the formation and subsequent removal of the circulating immune complexes from the bloodstream during the process of digestion

Determination of the scattering particles' size by means of DLS

The measurements were performed on a PLSS laser correlation spectrometer (INTOX MED LLC, Russia). We used a heterogeneous research scheme. The measurements were carried out in the band of 2,048 or 8,096 Hz. With these measurement parameters, the time taken to study one sample was about 15 min.

The particle sizes in the study samples were calculated using previously described software products included with the PLSS laser correlation spectrometer (QELS for Windows, Version 3.2, Division of Molecular and Radiation Biophysics, National Research Center “Kurchatov Institute” B.P.Konstantinov St Petersburg Nuclear Physics Institute, Russia)^30,33. The measurement result is presented as a histogram of the particle size distribution (histogram of the subfraction composition), in which the abscissa is the scale of dimensions in nanometers, and the contribution to the total scattering of a sample of particles of a given size in percent is plotted along the ordinate axis. In this case, the total scattering of all particles of the sample is taken as 100%. For each sample, measurements were taken at least three times. Statistical processing of the obtained data was carried out using the regularization algorithm³³ with the help of QELS 3.2 software, included in the set of the PLSS laser correlation spectrometer.

See Supplementary File 1 for more information.

Preparation of blood samples for analysis through the DLS method

In the experiment, 10 healthy men aged 24–40 years participated voluntarily. All volunteers were chosen from the staff of the Division of Molecular and Radiation Biophysics where the study was performed (National Research Center “Kurchatov Institute” B.P.Konstantinov St Petersburg Nuclear Physics Institute, Russia). All the participants conducted routine daily activities and were not treated with any medical substances for the period of the experiments. Subjects were excluded if they were taking medication during the period of the experiment. Recruitment of exclusively male volunteers was accidental, and it does not bear any significance in this study.

Use of biological material (plasma) was approved by the Ethical Committee of Polenov Russian Scienific Research Institute of Neurosurgery (Saint Petersburg, Russia) (23/H-18 of 23/10/2017). The Institute is authorized to perform research involving human participants according to guidelines from the Ministry of Healthcare of the Russian Federation (Order of the Ministry of Health of the Russian Federation “On the Ethics Committee of the Ministry of Health of the Russian Federation” of 10.07.2015 № 435n) that are in agreement with the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from all donors involved to participate in the study. All blood samples were de-identified.

Blood sampling (5 ml) was done before meals and at 1.5, 3.5, 5, 7, 12 and 14 h after eating. First venous blood withdrawal was performed at 8 A.M. from fasted participants. After that, volunteers were provided with hot three-course breakfast (vegetable salad, main course (either meat or fish with garnish), either tea or coffee with a cake). The acquisition of blood plasma was carried out by the standard method³⁴. As an anticoagulant, heparin was added at a final concentration of 50 U/ml.

Removal of blood cells was carried out by centrifugation at 1,500g for 15 min. The supernatant was collected and for the sedimentation of platelets and decay products of the cell elements, 1 ml was centrifuged for 30 min at 12,500g. The resulting plasma sample was then diluted three times with standard isotonic phosphate buffer (pH 7.2) containing 10 mM EDTA and 1 mM sodium azide.

Removal of ICs from blood plasma

To remove ICs from the plasma, we used immune-affinity sedimentation: an excess of protein A, immobilized on sepharose beads (Pasteur Research Institute of Epidemiology and Microbiology, Russia), was added to the blood plasma. Protein A is a protein isolated from the surface of the cell wall of Staphylococcus aureus, it binds the Igs of mammals well, particularly IgG³⁵.

After a 10-min incubation at room temperature, the immobilized protein A, together with the Igs bound thereto, were removed from the plasma by centrifugation of 1,000g for 10 min. The supernatant, which is a blood plasma, devoid of Igs (both in free form and in the composition of ICs), was diluted three times with phosphate buffer.

Preparation of the samples for the determination of the isotypic composition of the immunological component of IC

Monoclonal antibodies to human Igs of different isotypes were added to the plasma samples to determine the isotypic composition of the ICs formed during digestion. Adding antibodies to human Igs of different isotypes to the biological fluid results in the specific binding of ICs containing Igs of this isotype, and in the formation of even larger aggregates.

To the samples of filtered blood plasma (180 µl), prepared according to standard procedure^30,33, 20 μl of a solution of monoclonal murine antibodies to human Igs of different isotypes, both in pure form and in various combinations, were added (See section “Antibody details”). In particular, we used antibodies to human IgG1, IgG2, IgG3, IgG4 and IgA. Previously, the antibodies were diluted five times with standard isotonic phosphate buffer (pH 7.2) containing 10 mM EDTA and 1 mM sodium azide.

Antibody details

In our study, we used only monoclonal murine antibodies (MMA) to human Igs of different isotypes. All antibodies (Test kit “Diagnostic monoclonal antibodies for light and for heavy polypeptide chains of human immunoglobulins” produced according to Specifications 9398-001-11122195-08) were custom-produced for us by Polignost LLC (Saint Petersburg, Russia).

MMA to human IgA (item number: 1H9)³⁶.

Antibodies were raised in the hybridoma strain originated as the result of hybridization of myeloma PX 653A cells with splenocytes from inbred mice SJL/J immunized with the mixtures of purified myeloma proteins IgA of κ and λ types. Antibodies recognize the epitope of the α chain, and they bind effectively both with human IgA1 and IgA2. MMA were purified with affinity chromatography on protein G.

MMA to human IgG1 (item number: 2C11)³⁷.

Antibodies were raised in the hybridoma strain originated as the result of hybridization of myeloma PX 653A cells with splenocytes from inbred mice SJL/J immunized with the mixture of several human myeloma proteins IgG1. Antibodies were purified with affinity chromatography on protein G.

MMA to IgG2 (item number: 3C7).

MMA to IgG3 (item number: 5G12).

MMA to IgG4 (item number: 5C7).

The procedures of the latter three MMA productions were the same as described above with application of immunogens IgG2, IgG3 and IgG4, respectively.

Preparation of rat blood serum samples for analysis of food CICs

In this study, six healthy adult 250–280 g male Wister strain white albino rats (10–11 weeks old) (FGUP PLZH Rappolovo, Russia) were studied. All procedures for rat care and use were conducted in accordance with the National Standard of the Russian Federation GOST R 53434-2009: Principles of good laboratory practice (introduced 01.03.2010 by Federal Agency for Technical Regulation and Metrology, published by Standardinform, Moscow, 2010). The protocol was approved by scientific committee of the Division of Molecular and Radiation Biophysics of National Research Center "Kurchatov Institute" B.P.Konstantinov St Petersburg Nuclear Physics Institute (Gatchina, Russia).

Animals were individually housed in standard polycarbonate cages (width: 206 mm, depth: 365 mm, height: 197 mm) and maintained under standard conditions of temperature (22 #177; 2°C), humidity (55 ± 10%), artificial 12-hour light–dark cycle (lights on at 8:00 A.M., lights off at 8:00 Р.M.).

The rats were divided into two groups (n=3): those that received complete dietary rations for 3 days (group one) and those that did not receive any food at all (group two). For our tests, animals were allocated to groups in a random manner. The rats in group one were fed on the nutrient extruded and granulated food designed for experimental rodents (LLC) Laboratorkorm, Russia), and they had ad libitum access to it. All animals had ad libitum access to water.

All efforts were made to ameliorate any suffering of animals. Rats from both groups were anesthetized for the euthanasia with diethyl ether (PJSC “Medkhimprom”, Russia) through the respiratory route by exposing animals to the ether couples for approximately 2 min in an airtight transparent acrylic jar. Dead rats were opened, and their blood was sampled from the Vena portae and from the Vena cava inferior.

Blood was collected into the standard tubes with a clot activator and incubated at 37°C for 60 min, until the clot was completely formed. The clot was separated and the serum was centrifuged at 1,500g for 15 min. The supernatant was collected and again centrifuged for 30 min at 12,000g. The obtained serum sample was diluted three times with isotonic PBS (pH 7.2) containing 10 mM EDTA and 1 mM sodium azide. Each analysed serum sample was divided into two aliquots, one of which served as a control. To the other, 10% (v/v) of protein A, immobilized on sepharose microspheres (20 μl microspheres were added to 180 μl serum), was added.

Statistical analysis

Statistical processing of the obtained data was carried out using the regularization algorithm³³ through application of software QELS 3.2, included in the set of the PLSS laser correlation spectrometer (QELS for Windows, Version 3.2, National Research Center "Kurchatov Institute" B.P.Konstantinov St Petersburg Nuclear Physics Institute, Russia).

In each individual experiment, the error in determining the mean hydrodynamic size of the light-scattering particles in the study samples of biological fluids can be several tenths of a percent (and this value is reproduced when the experiment is repeated on the same sample). The error in determining the mean hydrodynamic size of the light-scattering particles on a series of equally prepared samples is usually higher by an order of magnitude.

To analyze the dynamic of accumulation of food ICs and their subsequent dissipation in blood serum samples from 10 volunteers, applying DLS, we calculated mean and standard deviation (SD) at several time points for the following parameters of interest: hydrodynamic radius of the scattering particles, their contribution to the light scattering. Mean inter-group difference was calculated with 99% confidence intervals.

The same calculations were carried out to evaluate isotypic composition of immunoglobulins of human CIC after eating.

Computations were performed using MS Excel 2007 standard statistical functions (Statistical Package Microsoft Office 97 for Windows, Redmond, USA).

Peculiarities of the formation and utilization of the CIC during digestion in rats

In the experiment, two groups of rats participated: the first group of animals received a complete food ration for 3 days and the second was starved. For the analysis by the DLS method, serum from the rat portal vein of the liver and from the inferior vena cava was used.

To reveal the CICs that formed during the process of digestion, we applied the approach associated with the selective removal of Igs and their complexes from the analysed blood sample using protein A immobilized on sepharose beads, followed by centrifugation^31,32. Comparison of the histograms of the subfractional composition of particles of blood serum samples, those processed with protein A and those not processed obtained by the DLS method, allows for the recording of the presence or absence of a fraction of large scattering particles, the hydrodynamic radius (R_h) of which corresponds to the size of ICs.

The results from analysis of the blood serum from the portal vein of rats that received a full-fledged food ration are shown in Figure 1. It can be seen that in the blood of the animal, there are large protein aggregates, with an R_h of the order of 200–300 nm (Figure 1A), disappearing after treatment of the serum with protein A immobilized on sepharose microspheres (Figure 1B). We have identified these protein aggregates as food CICs.

Figure 1.

Histograms of the particle distribution by the size of the blood serum samples from the portal vein of liver of the rat that received a wholesome food ration for 3 days (A and B) and the rat that starved for 3 days (С). The figure presents the results of analyses of the blood serum samples each taken from one most representative rat from each experimental group. (A, С) Samples of native serums. (B). Sample of serum, treated with protein A, immobilized on the agarose microspheres. The x-axis is the hydrodynamic radius of the particles in nm, the y-axis is the contribution to the scattering in %.

The portal vein in animals is a venous trunk, through which blood from the stomach and intestine passes into the liver. Thus, in the blood of a satiated rat that is coming from the digestive tract to the liver, a substantial amount of CICs are observed (Figure 1A). In the blood serum taken from the portal vein of the rats that were starved for three days, the CICs are practically absent (Figure 1С).

The results of research of the blood samples from the portal vein of the liver and from the inferior vena cava of the fed and hungry rats also differ substantially; this is evidenced by the data presented in Figure 2.

Figure 2.

Histograms of the particle size distribution of the serum samples of hungry rats (A and B) and rats receiving a wholesome food ration (С and D). Blood was taken from the portal vein of the liver (A and С) and the inferior vena cava (B and D). x-axis, the hydrodynamic radius of the particles in nm; y-axis, the contribution to the scattering in %.

The inferior vena cava collects venous blood from most of the abdominal and pelvic organs, the walls of these cavities and lower limbs. By comparing blood from the portal vein of the liver and inferior vena cava, it is possible to obtain the information about changes in blood composition due to the filtration processes performed by the liver.

Comparison of Figure 2A and B shows no changes in the blood serum particle subfractional composition of the rats that starved for 3 days, before and after filtration in the liver. The contribution to the scattering of the fraction of particles matching the ICs in the blood that enters the liver of their gastrointestinal tract (6.1%, Figure 2A) practically matches the analogous indicator in the blood after the passage through the liver (5.8%, Figure 2B) in the starving rats.

Contrary to that, the contribution to the scattering of particles with the R_h that matches the ICs in the blood sample, taken from the portal vein of the rats that received a normal food ration is higher by more than an order of a magnitude than the contribution to scattering of particles of the same size in a blood sample taken from the inferior vena cava (73.3%, Figure 2C versus 5.4%, Figure 2D).

Thus, the presented results allow us to state that in the process of digestion, a substantial number of ICs that diffuse into the blood are formed in the intestine of rats. These food ICs are completely utilized in the liver.

Peculiarities of the formation and utilization of the CICs during digestion in humans

In total, 10 healthy men participated in the experiment. Blood sampling was done before meals and at 1.5, 3.5, 5, 7, 12 and 14 h after eating.

We determined the total contribution to the scattering of the fraction of ICs and the isotypic composition of the Ig component of the food CICs. Analysis of the results showed that the level of ICs in all inspected donors gradually increase in the blood, reaching its maximum at 3.5 h after eating.

As seen in Figure 3, the base level of the contribution to the scattering of the fraction of particles matching the ICs in the serum was less than 10%. The maximum IC contribution to the scattering was observed after 3.5 h, when it reached almost 80%. Then, the IC fraction contribution of the scattering decreased, reaching the basal level 14 h after eating.

Figure 3. Dynamics of the accumulation of immune complexes in the blood of healthy young men after eating.

x-axis, time after eating; y-axis, contribution to the scattering of the fraction of food ICs in %.

Table 1 presents the subfractional composition of the particles (the fraction that matches the ICs) of the serum of all the examined donors before eating and at the point of the maximum accumulation of ICs after eating (after 3.5 h). Before meals, there is an insubstantial variation in the size of the recorded ICs and a more substantial difference in the contribution to the scattering of IC fractions in the donors. At 3.5 h after eating, when the level of food complexes in the blood reaches its maximum, the sizes of the complexes for different donors practically match.

Analysis of changes in the isotypic composition of the Ig component of the CICs during the process of digestion in humans

As reported previously, the addition of antibodies to Igs of different isotypes to the blood plasma results in the specific binding of ICs, containing Igs of this isotype, and the formation of even larger protein structures. The appearance of a new fraction of light-scattering particles with a large R_h on the histograms of the subfractional composition of particles, obtained by the DLS method, attests to the presence of this variation of Igs in the analysed ICs. According to the contribution to the scattering of these high-molecular-weight protein aggregates to the total contribution of all ICs, it is possible to determine the proportion of ICs containing Igs of this isotype^30,32.

Analysis of the isotypic composition of the Ig component of the CICs showed that before eating, IgG1 Igs are predominantly present in CICs of all of the examined donors. After 3.5 hours after ingestion, the isotypic composition of Igs in the IC expands (Figure 4) along with the complexes of IgG1, the number increases to 33.1 ± 1.6% (mean ± SD). (Figure 4B), an insignificant number of IgG3 complexes (4.1 ± 0.9%) appears (Figure 4C), and the content of IgA complexes becomes predominant (42.0 ± 0.8%) (Figure 4D).

The results of the isotypic composition of Igs of the blood ICs of all examined donors are presented in Table 2.

Thus, the process of digestion in humans is accompanied by the formation of the CICs, the level of which reaches a maximum value the blood after 3.5 h. Initially, ICs mainly contains the IgG1, then a set of Ig isotypes in the composition of food ICs becomes more diverse. After 3.5 h, a gradual decrease in the amount of food ICs in the blood begins, and 14 h after eating, their concentration drops to the values corresponding to the initial level (before eating).