Natural antibody - Biochemistry and functions

Abstract

Natural antibodies have been common knowledge in the scientific community for more than half a century. Initially disregarded, their functions have garnered a newfound interest recently. Natural antibodies are usually polyreactive IgM antibodies and are implicated in numerous physiologic and pathologic processes. Current research demonstrates they play a role in adaptive and innate immune responses, autoimmunity, and apoptosis. Evidence exists that they are involved in the modulation of neurodegenerative disorders and malignancy. Furthermore, natural antibodies have been implicated in ischemia reperfusion injury and atherosclerosis. As such the study of natural antibodies may provide new insight into normal physiologic processes whilst concurrently paving the road for a wide-range of possible therapeutic options.

1. Introduction

1.1. History of Natural Antibody

Natural antibody (nAb) has been known of by the scientific community for more than half a century. It is defined as a subset of immunoglobulins that are produced without prior stimulation by an antigen or pathogen. For instance, nAbs are present in animals grown in antigen free environments and can be isolated from cord blood. For a long period, nAb was merely regarded as insignificant background of immunity. However, an early study in 1932 indicated that nAb in normal serum could contain bacteria [1]. Ten years later, another study noted that nAb may bind to cellular components of normal and neoplastic cells [2]. In recent years, research on nAb has gained significant insights of its physiological and pathological functions.

1.2. Structure of Natural Antibody

NAb are classically known to be IgM antibodies that have a wide variety of specificities for self and non-self antigens in the absence of exposure to these antigens.

Natural IgM antibodies are pentameric, consist of five singular IgM molecules that are joined by a J chain at the Fc ends. This makes nAb an important activator of complement as studies have shown IgM to be incredibly more efficient at complement mediated cell lysis than IgG [3]. The complement fixing sites of IgM are located in Cμ4 domain in Fc end [4–7].

The repertoire of natural antibody is restricted. The VDJ gene segments of nAb are in germline coding comparing with the hypermutated antibodies generated through adaptive immunity. Because of the lack of somatic hypermutation, nAb binds antigen in low affinities [3]. Despite such a confined repertoire, nAb exhibit marked reactivity towards a multitude of antigenic epitopes such as nucleic acids, carbohydrates, phospholipids, and various proteins.

Not all nAbs are of the IgM isotype, however. IgG and IgA nAbs have also been found. For instance, natural occurred IgG antibodies are discovered to be involved with clearance of red blood cells [8]. IgE natural antibodies have also been isolated from placental blood [9].

1.3. Cellular Source of Natural Antibody

Natural antibodies are mostly produced by a particular subset of B lymphocytes, B1 cells. B1 cells differentiate during the fetal and neonatal period and are generally localized in the peritoneal and pleural cavities [3]. A recent study also shown that the nAb repertoire can be regenerated in liver and bone marrow with little B1 cells after widespread ablation of the lymphoid system [7]. This suggests the regeneration or generation of nAb may not entirely depend on B1 cells exclusively. The production of natural antibody by B1 cells is regulated, at least in part, by phosphoinositide 3-kinase p100 delta [10], and maybe by MHC alleles [11].

2. Physiologic Functions of Natural Antibody

2.1. First line defense system against infection

Natural antibodies play an important role in preventing infections. As the first line defense system, nAb aids the targeting of pathogens, e.g. virus, to secondary lymphoid organs, thus preventing or mitigating the dissemination of pathogens to vital organs [12].

Once pathogens enter the bloodstream, they may be detected by polyreactive nAb thus activating complement. Mice deficient in natural IgM had an increased mortality upon bacterial infection than wild type mice [3]. Furthermore, resistance to invasive bacterial disease was conferred from IgM purified from the serum of normal animals. IgM deficient mice also decreased the production of specific IgG, which is important for adaptive immune responses. Natural antibodies have been located in the mucosal surface of mice nasopharynx [14], which are able to bind and colonize the Haemophilus influenzae in the host’s nasopharynx. In humans, monoclonal natural IgM antibodies that are protective against pneumococcal septicemia have been isolated [13].

Besides its protective role against bacteria, nAb has been shown to neutralize virus [15]. The mechanism is through nAb mediated activation of complement, which leads to pathogen destruction by complement membrane-attack-complex. Immune complex may also form to facilitate the clearance of pathogens from the bloodstream. For instance, during the initial phase of infection of influenza virus, nAb mediates viral neutralization followed by activation of the classical complement pathway [16]. In addition, nAb is involved in long-term IgG production against influenza virus, thus producing a long-term antibody response [17]. Natural IgA antibodies also play an important role in HIV infection through specific recognition of HIV capsid protein [8,18], resulting in proteolysis of the virus.

2.2. Facilitate Apoptosis

Natural Ab have also been implicated in “housekeeping” by promoting the clearance of apoptotic cells and thus attenuate inflammatory responses [8]. Mice with a deficiency in IgM are unable to fully dispose of apoptotic cells, and the clearance of certain cells depends on IgM to activate the classical complement pathway. An antigenic epitope thus far discovered is a modified form of phosphorylcholine, which is exposed on apoptotic cells allowing detection and clearance by nAb.

Recently, data has emerged that supports the notion that natural antibodies may be involved in clearing other cellular components as well. For instance, natural occurred anti-Gal antibody is found in normal individuals and can bind alpha-gal liposomes to activate complement leading to the recruitment of macrophages and neutrophils [20].

2.3. Role in Neurodegenerative Disorders

It has been reported that nAb may play a role in Alzheimer’s disease. Like many other neurodegenerative disorders, Alzheimer’s disease is characterized in part by the accumulation of misfolded protein. Natural Ab against serum amyloid A protein and alpha-synuclein has been found in human [19], and appears to have a much greater affinity for the aggregated and fibrillar forms of these proteins than to the monomeric form [8]. Natural antibodies against the toxic variants of beta-amyloid decreased with age and with progression of Alzheimer’s disease [21]. In vitro experiments showed that these nAbs are able to prevent cell death in neuroblastoma cells. IgG antibodies isolated from healthy subjects or Alzheimer’s patients exhibited an ability to protect neurons from beta-amyloid toxicity. Pilot study utilizing IVIG, which is known to contain nAb, in patients with Alzheimer’s disease showed improvement in the patient’s mental score.

Human nAb that binds to areas of demyelination has been isolated [8], and is able to attenuate apoptosis and cause regression of the demyelination. In addition, human nAb was implicated in remyelination in mouse models, but the exact mechanism is still under investigation [22].

2.4. Role in Malignancy

Many different tumor specific monoclonal antibodies have been discovered and majorities of these are IgM in germ-line encoding sequences. They recognize cell surface carbohydrate and not peptide epitopes, and can induce apoptosis in these tumor cells [23].

One example of a tumor specific antibody is SAM-6, which was isolated from a patient with gastric cancer [23]. The epitope is a carbohydrate. Upon binding to malignant cells, SAM-6 causes an increase in intracellular lipid and triglycerides. It is believed that this results in lipotoxicity which activates caspases through cytochrome c release ultimately ending in apoptosis of the cell. This antibody was tested in a mouse model of human pancreas carcinoma. The analysis showed a decrease in tumor mass and the authors claimed higher levels of apoptosis in the antibody group versus control. Similar effects were noted by another tumor specific nAb, SC-1, which helped regression and apoptosis in primary stomach cancers [23]. It is important to note, however, that malignant cells may also exhibit defense mechanisms against natural antibodies. An example is the expression of cell surface membrane complement regulatory proteins which serve to prevent antibody mediated attack of the cancer cell [24].

Natural antibodies against the neuroblastoma cells have been purified from healthy donor and used in the treatment of children with relapsed neuroblastoma [25]. The pilot study showed some promising results and supported further studies.

An important concern in many cancers is metastasis. Anti-Gal natural antibodies have also been studied in tumor [26]. When alpha-gal glycolipids were introduced to tumor cells and incorporated into the cell membrane, anti-gal nAb is able to bind and activate of complement, which leads to prevent metastasis of melanoma in mice. In addition, nAbs against Nerve Growth Factor were able to reduce the metastasis of prostate cancer cells [27].

Not all nAbs are protective against tumor. In fact, certain nAb clones recognizing apoptotic cells are actually associated with poor clinical outcome in patients with Chronic Lymphocytic Leukemia [28].

2.5. Role in Atherosclerosis

Atherosclerosis involves a chronic inflammation. Recent studies have suggested a protective role for natural IgM antibodies in atherosclerosis [29]. An example is the IgM antibody known as T15/EO6, which showed protection against atherosclerosis. Several mechanisms have been proposed. One is that the IgM antibody may bind and prevent the activation of endothelial cells. Another possibility is that IgM antibodies which normally bind apoptotic epitopes also recognize the oxidation epitopes on oxidized LDL [29], and modified LDL particles can induce a strong IgM response during atherogenesis [30]. Thus, natural IgM may prevent the uptake of oxidized LDL by macrophages thus halting foam cell formation. Experiments in animal models showed that transferring anti-oxidized LDL antibody or active immunization with the epitope is able to attenuate the development of atherosclerosis, raising an interesting therapeutic possibility [31].

Researchers have also demonstrated that an apolipoprotein mimetic peptide, 4F, was able to increase the titers of nAbs to epitopes found as a result of oxidation [32], and such nAbs are able to prevent atherogenesis at earlier but not advanced stages. The mechanism of such nAb protection is through binding of complements and decrease of proinflammatory cytokines such as TNF-α and IL-6 [33].

Natural occurred anti-phosphorylcholine antibodies have recently gained some spotlight as a novel risk marker for development of ischemic stroke and myocardial infarction [34,35]. Low IgM anti- phosphorylcholine is reported to be associated with ischemic stroke and myocardial infarction.

3. Pathological Roles of Natural Antibody in Ischemia/Reperfusion Injury and transplantation

In addition to roles of nAb in normal physiology, nAb also contribute to pathological conditions. Recent studies showed that nAb plays an important role in ischemia-reperfusion injury (I/R) [36–39].

I/R injury is a significant complication of many clinical conditions, including myocardial infarction, cerebral ischemic events, intestinal ischemia, various conditions requiring vascular surgery, trauma, and transplantation [40].

Early studies have showed that an acute inflammatory response appears following tissue ischemia and involves complement system [41,42]. Newest evidence suggests that I/R injury is initiated by recognition and binding of natural IgM to newly expressed epitopes on hypoxic cells [43,44]. It is also reported that natural IgM antibodies recognize phospholipids as antigens in I/R injury [45].

Interestingly a study has examined whether a peptide that prevents IgM binding to neo-epitopes in mice would be efficacious in rats⁴⁶. The peptide was shown to prevent reperfusion injury in mice and was tested in rats. The rats were subjected to ischemia and then given the peptide prior to reperfusion. The data demonstrate that rats given the peptide exhibited milder signs of reperfusion injury such as less hemorrhage and edema as well as reduced signs of damage in microscopy [46]. The authors pointed out the potential use of this peptide in other species in light of its efficacy in rats.

Recent work has indicated that certain B-1 cells are responsible for producing pathogenic IgM [47]. Furthermore, an antigenic target for I/R specific natural IgM is a self-antigen, non-muscle myosin heavy chain II [44,48]. Another antigen, annexin IV, is also suggested as a target of the natural IgM mediated inflammation in I/R injury [49].

Natural Ab may also play a role in xenograft rejection, and a possible mechanism is that nAb binds to carbohydrate epitopes on the xeno-grafted cells thus leading to graft rejection [50].

4. Conclusions

Recent research on natural antibodies has revealed its multiple roles in physiology and pathology. Because of its limited repertoire, future research will define subsets that are either beneficial or potentially harmful to the homeostasis. This may help identify useful tools for detection of disease or targets for new therapies.