**3.5. RBC transport Tissue Specific Antigens (TSAs)**

If RBC transport antigens to central organs of the immune system to induce tolerance, then RBC will definitely transport TSAs. Otherwise this transport function has nothing to do with tolerance. Consequently, the objective of this experiment was to demonstrate that antibodies against TSAs can be prepared through injecting RBC of white mice into rabbits. Figure 3 illustrates the experiment.

**Figure 3.** Preparing antibodies against white mice TSAs in rabbit

## **Materials**

A number of white mice were slaughtered to collect their blood on sodium citrate and their organs (liver, kidney and spleen) were preserved on 10 % formalin. The separated RBC were Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Role in Health and Disease 19

washed many times with sodium citrate and then diluted with 3% formol-saline to kill any bacterial contamination. An ordinary rabbit was selected to prepare the antibodies.

#### **Method:**

18 Blood Cell – An Overview of Studies in Hematology

**3.4. RBC transport self HLA antigens** 

**3.5. RBC transport Tissue Specific Antigens (TSAs)** 

Prepare

Prepare

Add

**Figure 3.** Preparing antibodies against white mice TSAs in rabbit

Fluorescent Anti Rabbit Ig

as usual.

HLA antigens.

**Materials** 

female spouse hemolysate.

illustrates the experiment.

**Results** 

Male spouse lymphocytes was added and followed by the complement and eosin dye

It was observed that female spouse hemolysate inhibited the typing reaction while the third person hemolysate did not. This indicates the existence of male spouse HLA antigens in

This experiment is similar to the previous one. The only difference is the use of the male's own hemolysate instead of his female's spouse hemolysate. It was observed that a male hemolysate inhibited the typing reaction of his lymphocytes indicating the existence of self

If RBC transport antigens to central organs of the immune system to induce tolerance, then RBC will definitely transport TSAs. Otherwise this transport function has nothing to do with tolerance. Consequently, the objective of this experiment was to demonstrate that antibodies against TSAs can be prepared through injecting RBC of white mice into rabbits. Figure 3

**RBC**

A number of white mice were slaughtered to collect their blood on sodium citrate and their organs (liver, kidney and spleen) were preserved on 10 % formalin. The separated RBC were

**Mouse Tissue sections**

Apply

Inject

Antiserum

Wells that gave positive reaction in typing were examined by contrast microscope.


#### **Results:**

All sections showed florescence. Figure 4 illustrates some of the histopathology sections taken from a white mouse's organs.

**Figure 4.** Histopathology sections from a white mouse's organs examined by florescent microscope showing florescence due to antigen-antibody reaction, A: kidney tissue, B: liver, and C: spleen.

## **3.6. RBC hemolysate antigens are precipitated by plasma**

Ouchterlony immuno-precipitation test of normal serum against self and other normal hemolysate was conducted, Figure 5(a). We confirmed this finding by using Western Blot technique, and showed that serum from one individual recognized antigens in hemolysate from two normal persons, Figure 5(b). Further confirmation was obtained by using twodimensional gel electrophoresis (2-DE) of co-immunoprecipitated hemolysate antigens using self-serum, Figure 5(c). Notice that the number of the immune-precipitated antigens is numerous and many spots were enriched by immune-precipitation because those antigens were not detected in 2-DE gel of hemolysate, Figure 5(d). Antigenicity of the separated proteins was confirmed by immune-blotting proteins separated by 2-DE with the same selfserum, Figure 5(e). This excluded co-precipitation of non-antigens, as they would not be detected in immune-blotting.

## **3.7. RBC transport bacterial antigens**

As TB is a priority disease, trying to find Mycobacterium tuberculosis bacilli protein antigens (MTPAs) in TB-patient hemolysate was conducted through 2D electrophoresis, and

then identifying gel spots with mass spectrometry. Fortunately, we discovered four MTPAs. This motivated us to do the experiments of the next section to identify more MTPAs in hemolysate of TB patients.

Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Role in Health and Disease 21

**Figure 6.** Flowchart depicting the resources and steps for identification of hemolysate antigens related

 **Bacteria:** *Escherichia coli* O157:H7 strain1 was inoculated onto SMAC agar (Oxoid). Colonies were tested by *E. coli* O157 latex kit (Oxoid DR 620) and confirmed biochemically. A single colony of *E. coli* bacterial growth from the plate was inoculated into Brain heart infusion broth (Oxoid) and incubated overnight at 37 C and adjusted

The rabbit and sheep were tested serologically, to be negative, for *Escherichia coli* O157:H7.

1. Rabbits were vaccinated by Escherichia coli. Rabbits were injected subcutaneously with

1 This strain is kindly provided by the serology department, Animal Health Research Institute (AHRI), Giza, Egypt.

2. Blood was collected from the ear-vein after 21 days from the first injection.

**Figure 7.** Precipitated Antigens separated using 2D electrophoresis Gel

to a concentration of approximately 1010 CFU.

2. A baladi sheep between 8 to10 months.

one ml on weekly basis for three weeks.

The first experiment method was done as follows, Figure 8:

to *Mycobacterium tuberculosis* (H37Rv)

**Materials** 

**Animals:** 

**Methods:** 

1. A rabbit

#### **Identifying MTPAs in TB patients hemolysate**

The goal is to find the set of antigens, in TB patients' hemolysate, which is related to Mycobacterium tuberculosis bacilli. The approach taken follows the following steps Figure 6:

	- [A] Patients
	- [B] Mycobacterium tuberculosis (H37Rv)
	- Collect blood sample on anticoagulant (step 1)
	- Separate RBC and wash many times with saline (step 2)

**Figure 5.** Detection of innumerable antigens in Red Blood Cells. (a) Ouchterlony test showing serum of normal against hemolysate of self and others (b) Western Blot using two normal hemolysate propped with serum of one of them (c) Silver stain of 2-DE of immune-precipitated hemolysate antigens (d) Silver stain of 2D electrophoresis of hemolysate (e) Western Blot of 2-DE of hemolysate propped with serum.

## **3.8. RBC antigens and plasma antibodies concentration vary with time**

The objective of this experiment is to investigate the dynamics of foreign antigens in RBC. In effect, antibodies are taken at one instance of time, while RBC are taken at different instances.

Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Role in Health and Disease 21

**Figure 6.** Flowchart depicting the resources and steps for identification of hemolysate antigens related to *Mycobacterium tuberculosis* (H37Rv)

**Figure 7.** Precipitated Antigens separated using 2D electrophoresis Gel

#### **Materials**

20 Blood Cell – An Overview of Studies in Hematology

**Identifying MTPAs in TB patients hemolysate** 

[B] Mycobacterium tuberculosis (H37Rv)

binding buffer in affinity chromatography

TOF mass spectrometry (step 6)

Collect blood sample on anticoagulant (step 1)

4. Prepare hyper immune serum for M. *tuberculosis* (step 3) 5. Purify antibodies using Protein A Sepharose beads (step 4)

Separate RBC and wash many times with saline (step 2)

hemolysate of TB patients.

1. The study resources are: [A] Patients

2. For each patient:

Figure 6:

with serum.

instances.

then identifying gel spots with mass spectrometry. Fortunately, we discovered four MTPAs. This motivated us to do the experiments of the next section to identify more MTPAs in

The goal is to find the set of antigens, in TB patients' hemolysate, which is related to Mycobacterium tuberculosis bacilli. The approach taken follows the following steps

3. Hemolysate [C] is prepared by rupturing RBC with low isotonic solution which is the

6. The purified antibodies are then used to separate antigens from hemolysate (step 5) 7. The disease related antigens are identified using in gel trypsin digestion and MALDI

**Figure 5.** Detection of innumerable antigens in Red Blood Cells. (a) Ouchterlony test showing serum of normal against hemolysate of self and others (b) Western Blot using two normal hemolysate propped with serum of one of them (c) Silver stain of 2-DE of immune-precipitated hemolysate antigens (d) Silver stain of 2D electrophoresis of hemolysate (e) Western Blot of 2-DE of hemolysate propped

The objective of this experiment is to investigate the dynamics of foreign antigens in RBC. In effect, antibodies are taken at one instance of time, while RBC are taken at different

**3.8. RBC antigens and plasma antibodies concentration vary with time** 

	- 1. A rabbit
	- 2. A baladi sheep between 8 to10 months.

The rabbit and sheep were tested serologically, to be negative, for *Escherichia coli* O157:H7.

#### **Methods:**

The first experiment method was done as follows, Figure 8:


<sup>1</sup> This strain is kindly provided by the serology department, Animal Health Research Institute (AHRI), Giza, Egypt.

3. Rabbits sera were separated and examined for antibodies against E. coli O157 using direct bacteria slide agglutination test.

Rediscovering Red Blood Cells: Revealing Their Dynamic Antigens Store and Its Role in Health and Disease 23

The RBC transport function maintains tolerance to self antigens. This function is exploited positively to protect a fetus from the immune system attack using the same mechanism of

In humans and animals, not all microorganisms are capable of causing disease. Some of those microorganisms are equipped with the machinery that can overcome biological barriers and can cause disease in animals but not in humans and vice versa [15]. The role of RBC antigens transport in inducing tolerance to self-antigens is a feature that can be considered as a security-hole, as invaders can exploit this process to escape from the response of the immune system by disguising themselves as self. Tumors and parasites are

Notice that this mechanism of tolerance induction does not contradict with all what we know about tolerance. Further, it explains the documented properties of tolerance. For

 Artificially induced tolerance is of finite duration because antigen stores get depleted. Tolerance to self antigens is a process that continues throughout life but begins during

Notice that the discovered function of RBC fills a gap in the understanding of tolerance. Part

1. Why soluble antigens administered intravenously favor tolerance while particulate

2. Why ingested large doses of soluble proteins induce systemic T lymphocyte tolerance, whereas the components of vaccines such as the Sabin polio vaccine induce an effective

Answer of Question 1 and 2: RBC can easily absorb soluble antigens through pinocytosis while a particulate antigen needs receptor sites on RBC in order to be absorbed, which is the RBC membrane antigens function. Notice that the probability that the immune system will react to some processed antigens still exists. That is why the dose of antigens plays an important role. As far as there are enough stores of antigens in RBC, they are effectively

Answer of Question 3: If antigens are introduced to a fetus while the immune system is still incapable of respond, there is a good chance for those antigens to be processed by the Antigen-Presenting-Cells (APC) and then absorbed by RBC. When mature lymphocytes production starts, later in life, antigen stores of RBC are used to induce tolerance. This may

Further, a pathogenesis mechanism of some autoimmune disease can be postulated. If RBC antigen-transport function is impaired for a particular self-antigen, for some reason, the

fetal development because RBC are transporting self antigens all the time.

3. Why tolerance is easier to induce in prenatal rather than postnatal life.

protecting the self. In effect, a fetus, which is an allograft, is considered part of self.

**4. The role of RBC antigens transport in health and disease** 

instance, some of the properties that can be explained are:

of this gap can be expressed in the following questions:

antigens injected into the skin favor immunity.

explain why tolerance is easier to induce in prenatal life.

local immune response.

tolerated.

negative examples.


#### **Results**

The rabbit serum showed high titer (1/160) of antibodies against E. coli. Antigens of E. coli could be precipitated from sheep RBC of the 1st and 2nd week after infection, only, Figure 9.

**Figure 8.** Preparing Antibodies against bacteria and preparing RBC carrying antigens of this bacteria. The purpose is to precipitate Bacteria antigens from RBC of infected animals using the prepared antibodies against those antigens.

**Central well:** Rabbit antiserum **Well 1:** Sheep's RBC before infection **Well 2:** Sheep's RBC 1st week after infection **Well 3:** Sheep's RBC 2nd week after infection **Well 4:** Sheep's RBC 3rd week after infection

**Figure 9.** Illustrates the dynamics of RBC's antigens
