The Plasma Preincubation with Papain Before the Assay Suggests that a Gell and Coombs Type II Reaction is Been Demonstrated by the Leukocyte Adherence Inhibition Test

Background: The Leukocyte Adherence Inhibition Test (LAIT) is an ex vivo challenge test used to testimony specific immunoreactivity. Despite numerous studies had been performed to elucidate the interactions involved in the assay, there are yet several questions about its mechanisms and how to translate its results into the medical practice. Objectives: To perform an experiment with an antibody disassembler: the papain, to demonstrate the participation of specific antibodies in the reconnaissance phase of the assay performed with protein antigens. Methods: Paired side-by-side ex vivo allergen-specific challenges monitored by the LAIT, were performed with the plasma of allergic patients, preincubated (or not) with papain. Results: The mean of the differences (26.66%) of the two arms was considered significative by the paired t-test (p-value < 0.05). Conclusion: Our results suggests that the leukocyte adherence inhibition phenomenon and its inhibition is possibly mediated by an interaction of cells and specific antibodies and deserves further studies as an ex vivo challenge test tool for research of Gell & Coombs type II hypersensitivity reactions. dos Santos. The Plasma Preincubation with Papain Before the Assay Suggests that a Gell and Coombs Type II Reaction is Been Demonstrated by the Leukocyte Adher ence Inhibition Test. Biomed J Sci & Tech Res 36(3)-2021. BJSTR. MS.ID.005869.


Introduction
Leukocytes are immune cells that play a key role in vertebrate physiology. It is their capacity of adherence and mobilization that protects the host providing inflammation, expelling of foreign bodies and killing of microorganisms in order to allow cicatrization of injured tissues [1]. Unfortunately, their disfunction, whether by hyperactivity or inactivity, also produces diseases [2]. This is easy to understand because allergic responses are amplified cascade inflammatory reactions that rely on multiples sequential steps, as well on the control of several modulators [3]. To mobilize defenses, leukocytes depend on cytokines, antibodies, surface receptors and adhesion molecules [4]. Adhesion molecules are cytokineregulated glycoproteins expressed on the surface of leukocytes that mediate the adhesion of immune cells to foreign bodies and regulate the recruitment and migration of circulating cells through compromised tissues towards the inflamed site [5][6][7]. The inhibition of the leukocyte capability of migration induced by specific antigens (ex vivo challenges) was first studied by Boyden, who designed a migration chamber specially to perform the Leukocyte Migration Inhibition Test (LMI test) [8]. Within the same perspective, to study the inhibition of leukocyte adherence, following ex vivo challenges with specific antigens, Halliday designed the Leukocyte Adherence Inhibition Test (LAIT) [9]. Leukocyte's migration and adhesion are intimately related since the inhibition of adhesion also paralysis migration [10]. This interrelationship explains the equivalence that correlates the immunoassays comparing the inhibition of migration with the inhibition of adhesion of leukocytes [11].
Live and rest leukocytes in physiologic conditions have the natural capacity to adhere to glass, property easily recognized by an artisanal immunoassay done with help of a glass surface of a tube and/or a hemocytometer chamber, an optical microscope and a laboratory water bath [12]. This general capacity, however, is lost when the leukocytes are functionally engaged with a particular antigen [13]. When activated in the presence of a specific antigen, leukocytes release soluble factors (cytokines) that recruit nearby leukocytes abolishing their natural capacity of glass adherence and migration [14]. The quantification of the antigen-specific inhibition of leukocyte adherence and/or migration are/is evaluated by the comparison of an antigen-challenged assay with a concomitant control assay done with the unchallenged plasma [15]. Since several mechanisms have been implied as participants of these phenomena, the failure to migrate and/or to adhere to glass seems to be just the final indicator of the antigen-specific leukocyte activation characterized by the cytokines liberation or, to describe with a single word: the immunoreactivity [16][17][18][19].
After the initial studies of Halliday and co-works, several scientists evaluated the ex vivo challenges monitored by the LAIT and/or the LMI test to demonstrated immunoreactivity to specific antigens, in order to understand the role of these antigens in health and disease [20][21][22][23][24][25]. The studies were also directed to identify the specific cytokines involved in the inhibition of the adherence/ migration phenomena, as well their analytical interferents and pharmacological inhibitors [26][27][28]. But the liberation of cytokines is just the consequent response of the reconnaissance of the specific antigen. We, particularly, were intrigued to respond the question: "Is this cell-mediated phenomenon initiated by specific antibodies?" Whatsoever, the specificity and the prompt response to respond to challenging antigens suggests that an adaptive immune mechanism is involved with the presence of free and/ or membrane-bound specific antibodies. As we observed, it's enough just 30 minutes in contact with the specific antigen at an agitation device at physiologic temperature to generate the specific inhibition of the leukocyte adherence and/or the migration [29].
The involvement of immune cells and antibodies in the context of a hypersensitivity disease characterizes the general definition of a type II Gell and Coombs' reaction. The so-called type II Gell and Coombs' hypersensitivity reactions includes a wide variety of immune mechanisms that possess in common the participation of immune cells and antibodies such as IgG and/or IgM [30].
Usually the Gell and Coombs' type I reaction is defined by the interaction of IgE and mast cells, but now a days we known that IgE can also binds and interacts with circulant immune cells, such as neutrophils, lymphocytes, monocytes, basophils and eosinophils and, at least conceptually, is able to participate of type II reactions [31][32][33][34][35]. Despite the original description of Gell and Coombs had generally named the type II reaction as "cytotoxic", theirs subsequent textbook explanation recognized that some of these reactions were not necessarily deleterious to tissue, but could instead have a stimulatory effect inducing immune modulation or a blast transformation [30]. For a long time, the type II reaction was called "Antibody-Dependent Cellular Cytotoxicity" (ADCC), however, modern classification of hypersensitivity reactions divides type II reactions between type IIa (Antibody-mediated cytotoxic reactions) and type IIb (Antibody-mediated cellstimulating reactions) [36].  [37]. Papain promotes the endolytic hydrolysis of proteins with broad specificity for peptide bonds, especially amino acids bearing a large hydrophobic side chain at the second position at the cleavage site [38]. Papain acts by a catalytic dyad mechanism involving a nucleophilic cysteine thiol [39].
Since the early fifties, when Nobel laureated scientists, such as Dr. Linus Pauling were yet struggling to comprehend the biochemical structure of polypeptide chains and proteins, it was already known, mainly by Porter's experiments, that the hydrolysis of antibodies by papain results not only in the inactivity of the antibody, but produced also an inhibitory factor that competes by their specific antigen with the correlated untreated antibodies [40][41][42]. This antibody-inhibitory fraction, obtained by hydrolysis of human immunoglobulins by means of papain, was studied independently by several investigators and referred by diverse names until 1964 when a committee of the WHO recommended the use of the nomenclature: Antigen-Binding Fragment (Fab) to refer to the inhibitory fragments produced by papain [43,44]. Papain cleaves the upper hinge of antibodies, producing three fragments: two 50 KDa Fab fragments and one 50 KDa crystallizable fragment (Fc), losing its capacity of agglutination, precipitation, opsonization and lysis [45][46][47].
To evaluate the possibility that the initial antigen-specific reconnaissance phenomena observed in the ex vivo challenge of the LAIT is mediated by antibodies, we designed a study comparing, side-by-side, the performance of LAIT with the paired same samples of plasma preincubated (or not) with papain before the antigen-specific ex vivo challenge test. The hypothesis was that a partial or total loss of the capacity to inhibit the leukocyte adherence produced by specific antigen, observed after the papain treatment, suggests that the initial reconnaissance phenomenon is mediated by specific antibodies. The mechanism could be just the cleavage of the specific free or cell-bounded antibodies and/or the production of autologous Fab fragments able to compete by the antigen's epitopes.

Study Design and Methods
After receiving institutional review board approval, 11 consecutives outpatients with clinical symptoms of allergy, diagnosed by allergy-skin tests as previously described, to  Transglutaminase was gently provided by Ajinomoto Co., Inc.
(Limeira -SP -Brazil) to perform bovine beta-lactoglobulin (Bos D 5) polymerization, as previously described [29]. Plasma samples were collected in heparinized collection tubes. The duplicated plasma samples (100μL) were incubated (or not) during 30 minutes with papain (10μL, 1 mg/mL, 37 °C) before the ex vivo challenge with the specific antigen. The ex vivo challenge tests were performed as described previously, with minor alterations to fit the experiment with papain preincubation [29]. The plasma To further calculated the Leukocyte Adherence Inhibition (LAI) it is enough to subtract the LAR from 100 (%).

Statistical Analyses
Statistical analyses were performed using GraphPad Prism software (version 5.0; GraphPad Software, Inc., San Diego, CA, USA). The data were reported as arithmetic means with 95% confidence intervals (CI) and standard deviations (SD). Differences in the means of matched samples were assessed by paired t-tests. For all analyses, a p-value of less than 0.05 was considered significant. A whiskers-and-box plot graph was generated by the software.

Results
The

Discussion
The complexity of the Gell and Coombs type II reactions is with another occasional innate reconnaissance system, but the high efficiency in blocking the inhibition of the adherence suggests that this mechanism is been activated by antigen-specific adaptive immune response.
Another inevitable visualization of the experiment is the concept of "effector and blocking antibodies" [56]. Operationally we can classify the antibodies according with their main function in two categories: the blocking antibodies and the effector antibodies.  [60].
Despite the free serum IgE correlates well with allergic disease, there are some allergic patients that present normal IgE serum levels at immunoassays. That is why it is actually proposed the measuring, by the employ of lactic acid on plasma, of the liberated cell bounded IgE as a tool to help physicians to diagnosis allergy [61]. This may happen when the allergy is produced by a non-IgEmediated mechanism, or even when the serum presence of non-IgE antibodies interferes with the IgE immunoassay, competing by the allergens' epitopes [62]. Finally, we must also consider the Gell and Coombs' type III hypersensitivity reaction that involves the bounding of free antibodies to circulating antigens that produces Complement-mediated reactions when larger immunocomplexes reach the tissues. This is one of the many "deserved-to-be-studied" mechanisms that could also possibly be "Complementing" the cellular phenomena observed on the LAIT positivity or, at least, on the regulation of the immune humoral activity by the Complement System [63]. We theorize that the ex vivo tests to detect Complement activation, theoretically, could be a valuable tool to achieve this objective if performed in association with the LAIT [64]. Because papain is known to cleave not only immunoglobulins but also other proteins, we cannot conclude from our results that a Gell and

Declaration of All Sources of Funding
Olivier CE, Pinto DG, Lima RPS and Santos RAPG received grant support from Instituto Alergoimuno de Americana.

Disclosure of Potential Conflict of Interest
The authors have declared that they have no conflict of interest.