Natural Polyreactive IgA and IgM Autoantibodies in Human Colostrum

Secretory antibodies against bacteria and viruses in human colostrum and milk are known to be important protective factors for the breast-fed infant. The authors have shown by enzyme immunoassay that colostrum contains IgA and IgM antibodies to a number of autoantigens: native DNA, actin, myosin, myoglobin, laminin, transferrin and thyroglobulin. These antibodies were polyspecific—those with anti-DNA reactivity immunopurified on a DNA-cellulose affinity column bound to a panel of self- and environmental antigens. The levels of natural autoantibodies in the immunoglobulin fraction of human colostrum were 3–10 times lower (when presented as antibody activity per μg of immunoglobulin) than in the immunoglobulin fraction of serum. The biological significance of the presence of B cells with autoantibody specificity in the mammary gland and of natural autoantibodies in colostrum and milk is not clear. It has been suggested that self-reacting autoantibodies in serum play a major role in the selection of the pre-immune B-cell repertoire and in the maintenance of the immune homeostasis. The authors hypothesize that the natural autoantibodies in colostrum and milk may contribute to the selection process of physiological repertoire during the early postnatal period in breast-fed infants. This could explain the lower frequency of allergic, inflammatory and autoimmune diseases and lymphomas which is seen in their later life when compared with that observed in children who have been formula-fed after birth.     

Infectious Serologies and Autoantibodies in Hepatitis C and Autoimmune Disease-Associated Mixed Cryoglobulinemia

Mixed cryoglobulinemia (MC) syndrome is an immune complex-mediated vasculitis characterized by the clinical triad of purpura, weakness, and arthralgias, the morbidity of which is mainly related to kidney and peripheral nervous system dysfunction as well as to the development of a secondary lymphoma (Ferri et al. Autoimmun Rev 7:114?C120, 2007, Lidar et al. Ann N Y Acad Sci 1173:649?C657, 2009, Trejo et al. Semin Arthritis Rheum 33:19?C28, 2003). MC is associated with infectious and systemic disorders, principally autoimmune and lymphoproliferative diseases. Since the 1990s, a striking association (>90%) between MC and hepatitis C virus (HCV) infection has been established (Ferri and Bombardieri 2004; Pascual et al. J Infect Dis 162:569?C570, 1990). However, information regarding the etiopathogenesis of HCV-negative MC is scant (Mascia et al. Dig Liver Dis 39:61?C64, 2007). We hereby present our findings, as well as previously published data, regarding the presence of antibodies against infectious agents and autoantibodies in patients with MC in an attempt to establish novel associations which may shed light on the etiopathogenesis of this disease.     

The Significance of Natural Autoantibodies

Since Burnet first introduced his “forbidden clones” theory, the discrimination between self and non-self and the physiologic mechanisms of avoiding autoimmunity remained an enigma. The realization in the past two decades that autoantibodies reacting with various self antigens are common in normals has led to intensive research on the origin and physiologic role of these “natural autoantibodies”. After reviewing the extensive literature on the appearance of natural autoantibodies in normal animals and humans, and the studies proving unequivocally that natural autoantibodies are coded by gern line genes, we will discuss the current hypotheses explaining their appearance and physiologic role. Despite the fact that numerous hypotheses explaining the origin of natural autoantibodies have been postulated only the two important ones will be discussed. The first, proposed by Cunningham, holds that clonal deletion as viewed by Burnet operates in early life; however, later in life all autoreactive B cells not eliminated, during ontogeny are prevented from expanding and secreting anti-self antibodies by a compensatory suppressor mechanism. Therefore, natural autoantibodies are postulated to be autoantibodies which are produced only in minute quantities allowed by the suppressor mechanism. The second hypothesis views autoantibody formation as a result of cross reaction between foreign and self determinants. It is suggested that the part of the B cell population which gives rise to autoantibodies carries a polyspecific receptor; fixation of a foreign antigen to this receptor induces the B cell to undergo a series of divisions and mutations, which under the selective pressure of the antigen leads to production of a highly specific antibody. Thus natural autoantibodies may constitute the antibodies secreted by these B cells prior to encountering foreign antigens. The biologic role of natural autoantibodies is also elusive. The common denominator to all the theories dealing with that puzzling question is the view that natural autoantibodies have a positive role in normal immune reactions, perhaps even an essential role without which normal immune function would be disrupted. Grabar suggested that natural autoantibodies are part of a physiologic mechsnism for cleansing the organism of self and non-self products in which classical antibodies serve to clear the body of foreign invading agents, while natural autoantibodies rid the organism of its own catabolic products. Cohen and Cooke suggested that natural autoantibodies, by binding to self antigens, act as a filter preventing powerful immune response against self triggered by self mimicking determinants on foreign invading microorganisms. Others have suggested a role for natural autoantibodies in the idiotypic network. We propose a different function for natural autoantibodies, namely enhancing host immune reactions to foreign infectious agents, in a similar manner in which HLA antigens participate in the activation of B and T cells, The question of the origin and biologic role of natural autoantibodies is not a purely academic one, and understanding these mechanisms will certainly clarify the pathogenesis of autoimmune diseases and autoimmunity in general.     

Human Autoantibodies Recognizing Human and Mouse Preimplantation Stages

PROBLEM: To find out whether autoantibodies against human preimplantation stages are present in some human sera and, if so, whether the antibodies could be capable to affect the egg development and/or to trigger an activation of the complement system at the procedures of assisted conception. METHODS: 1. Immunohistochemistry on blots of human preimplantation stages. 2. Immunohistochemistry on paraffin sections of human and mouse preimplantation stages. 3. Culture of mouse morulae to analyze complement activation. RESULTS: 1. Some human sera contained autoantibodies against human preimplantation stages. 2. Human-mouse cross-reacting antibodies against preimplantation stages occurred. 3. Immune complexes, formed on mouse preimplantation stages, activated the complement systems in egg cultures, resulting in a damaging of the eggs. CONCLUSION: The presence of natural autoantibodies to preimplantation stages may be associated with reproduction failure, caused by a direct effect by the autoantibodies and/or an activation of the uterine complement system by the immune complexes formed.     

Natural Human Antibodies Retrieved by Phage Display Libraries from Healthy Donors: Polyreactivity and Recognition of Human Immunodeficiency Virus Type 1 gp120 Epitopes

We have characterized the human natural antibody repertoire that contains antibodies recognizing the human immunodeficiency virus type 1 (HIV-1) gp120. A panel of monovalent antigen-binding fragments (Fab) selected from IgM and IgG isotype libraries generated from peripheral blood mononuclear cells (PBMC) of a healthy, HIV-1 noninfected individual was analysed, reflecting that only IgM, but not IgG, Fab were able to recognize HIV-1 gp120. The IgM Fab antibodies were not restricted to any particular heavy chain variable region (VH) germ line gene. However, the recognition of gp120 is associated to polyreactive antibodies and all display low-affinity interaction. This correlates with the absence of any maturation process as somatic mutation or isotype switch as the nucleotide sequence analysis of the variable regions reveals they are expressed near to germline configuration. In addition, none of the antibodies showed any neutralizing activity on HIV-1-infected lymphocytes, reflecting that the natural anti-gp120 repertoire is not sufficient to neutralize HIV infection.     

VH Genes of Human Autoantibodies

Important questions about pathogenic autoantibodies are whether they arise from germline V genes that contribute to the immunoglobulin repertoire in normal persons, whether they stem from “abnormal” V genes that are peculiar to patients with autoimmune diseases, and whether they are encoded by unmodified or somatically mutated immunoglobulin genes. These issues influence the way we think about three aspects of autoimmunity: genetic susceptibility to autoimmunization, the origins of lesion-producing autoantibodies, and the prevention and medical management of autoimmune disorders.     

Sedimentation Characteristics of Human Thyroid Autoantibodies

Forty-six sera from patients with Hashimoto's thyroiditis were separated into 7S and 19S fractions by zone ultracentrifugation over sucrose density gradients. In each case at least 98 per cent of the anti-thyroglobulin activity was recovered in the 7S fraction. This was confirmed by chromatography on DEAE-cellulose. This also revealed significant antibody activity in a 7S γ-globulin fraction of fast electrophoretic mobility obtained from one serum giving a `clear' line on reaction with thyroglobulin in agar gel. Complement-fixing antibodies reacting with the thyroid microsomal fraction were also of low molecular weight. Thyroglobulin autoantibodies induced experimentally in rats were confined to the 7S fraction of serum. Initially, rabbits respond to the intravenous injection of human thyroglobulin with the production of 19S antibodies but by the 15th day, predominantly 7S antibodies are found.     

Polyreactivity of human monoclonal antibodies: Human IgM anti-rhesus monoclonal antibodies are frequently polyreactive

The aim of this study was to characterize human anti-Rhesus monoclonal antibodies cross-reacting with tissue antigens. Of the 155 monoclonal alloantibodies tested, 49 also reacted with intracellular antigens, as demonstrated by immunofluorescence assay on cryostat sections of animal and human tissues. This cross-reactivity was mainly a property of monoclonal alloantibodies belonging to the IgM isotype (among the 49 cross-reacting Mabs, 37 were IgM). The results confirm that during an immune response against a foreign antigen (alloantigen), B cells that produce polyreactive antibodies are not excluded from the pool of responding cells.     

The Antibody Repertoire of Early Human B Cells I. High Frequency of Autoreactivity and Polyreactivity

Cord blood and fetal liver B cells were immortalized using Epstein-Barr virus, and IgM antibodies from the resulting lines and clones were examined for their binding to a variety of auto-antigens and micro-organisms by ELISA and fluorescence assays. Auto-antigens tested included Fc of IgG, ssDNA and dsDNA, cardiolipin, histones 1-4, collagens type I and II, thyroglobulin, cytoskeletal components, and a tissue section screen. Of 71 cell lines tested, all but 19 showed some autoreactivity. All 32 fetal liver lines reacted to some self-antigens. In cord blood clones, 16 out of 26 bound to auto-antigens. Many of the clones reacted with more than one auto-antigen and were 'polyreactive'. Some of the cord blood clones bound to extracts of micro-organisms, showing specificity for both endogenous and exogenous antigens. The high frequency of CD5+ B cells in the cord blood (greater than 50%) and fetal liver (greater than 70%) argues for many of these clones being derived from this subset. Therefore, our data support the concept that many 'early' B cells produce polyreactive IgM which can bind to a variety of different auto-antigens and micro-organisms. These IgM antibodies are similar to those described by others as 'natural antibodies'.     

Binding Affinity of Human Autoantibodies: Studies of Cryoglobulin IgM Rheumatoid Factors and IgG Autoantibodies to Albumin

The binding affinity of cryoglobulin IgM rheumatoid factors (RF) for human IgG and of human IgG anti-albumin autoantibodies for HSA was measured by the molecular sieving technique. The binding affinities of the two autoantibodies were consistently low (10⁴-10⁵ I/M) as compared to the affinities of corresponding hyperimmune animal antibodies (10⁶-10⁸ I/M). The findings were discussed in relation to theories on human autoimmunity. The existence of strict autotolerance at the T cell level and of autoreactivity at the low affinity B cell level was considered to be best compatible with the findings of this study and with the major known facts of autoimmunity.     

Characterization of Autoantibodies to Natural Killer Cells in HIV-Infected Patients

Natural killer (NK) cells in HIV-infected patients have a reduced ability to generate non-MHC restricted cytotoxicity to a variety of target cells. The authors investigated antibodies to NK cells in HIV-infected patients and evaluated effects of these antibodies to NK cell numbers and function. Antibodies to NK cells were determined in 160 HIV-infected patients and 35 healthy controls. Flow cytometric whole blood methods were developed to detect antibodies to NK cells. Antibodies to asialo-GM1 were detected by TLC immunostaining. The presence of antibodies to NK cells was demonstrated in plasma of about one-third (54/160) of HIV-infected patients but rarely in controls (2/35). Autoantibodies bound to NK cells in vivo and were detected by a strong increase of surface immunoglobulin (Ig) on NK cells of HIV-infected patients. Anti-NK cell antibodies were warmreactive antibodies rather of IgG than of IgM phenotype. The prevalence of specific antibodies to asialo-GM1 was low (12.5%). Numbers of circulating NK cells did not differ significantly between antibody positive (99.5/μl) and antibody negative (141/μl) patients ( P  = 0.3). However, pre-incubation of healthy donors' NK cells with autoantibody positive plasma significantly inhibited cytotoxicity to K562 leukaemic cells ( P  = 0.002). Autoantibodies to NK cells in HIV-infected patients are present in the plasma of one-third of HIV-infected patients and are bound to NK cells in vivo . There is evidence that these autoantibodies can induce NK cell defects similar to those seen in vivo     

Human Natural Killer cell expression of ULBP2 is associated with a mature functional phenotype

NKG2D is an important activating receptor expressed on NK cells. Ligands (termed NKG2DL) for this receptor include ULBP1-6, MICA and MICB in humans; they are upregulated in stressed, cancerous or infected cells where they engage NKG2D to induce NK cell cytotoxicity and cytokine production.Expression of NKG2DL on effector cells has been described in mice and more recently in human cells. We confirm that NK cell lines and IL-2 stimulated primary human NK cells also express the NKG2DL, ULBP2. However, expression of ULBP2 was not a result of transfer from a non-NK cell to an NK cell and in contrast to recent reports we saw no evidence that ULBP2 expression targeted these NK cells for fratricide or for cytotoxicity by NKG2D-expressing, non-NK effector cells.ULBP2 expression was however linked to expression of mature CD57⁺ NK cells. In particular, expression of ULBP2 was strongest on those NK cells that had evidence of recent activation and proliferation. We suggest that ULBP2 could be used to identify recently activated “mature” NK cells. Defining this phenotype would be useful for understanding the ontogeny on human NK cells.     

Isolation and Characterization of Human Monoclonal Autoantibodies to Glutamic Acid Decarboxylase

Production of human monoclonal autoantibodies to glutamic acid decarboxylase M_r 65,000 (GAD₆₅), characterization of their isotype, binding affinity, V region sequences and competition with autoantibodies in patients' sera is described. Lymphocytes from a patient with Addison's disease who had GAD₆₅ autoantibodies without diabetes were immortalised and fused to a mouse/human hybridoma. In addition, mouse monoclonal antibodies to GAD₆₅ were produced using standard techniques. F(ab')₂s from our monoclonals and the GAD6 mouse monoclonal were used in competition with intact monoclonals and sera from diabetic patients for binding to ¹²⁵I-labelled GAD₆₅ (amino acids 46-586). Reactivities of the human monoclonals with GAD 65,000/67,000 M_r chimeras were also studied. Variable region genes of human monoclonals were sequenced and analysed. The human monoclonals (n = 3) had affinity constants for GAD₆₅ of 2.2 &#50 10⁹, 5.8 &#50 10⁹, 1.3 &#50 10¹⁰ mol/l^{&#109 1}; affinities of the mouse monoclonals (n = 5) ranged from 1.1 &#50 10⁸ to 5.4 &#50 10¹⁰ mol/l^{&#109 1}. The binding of each of the human monoclonals was inhibited by GAD6 F(ab')₂ and the binding of GAD6 antibody was inhibited by the human monoclonal F(ab')₂s suggesting that the epitopes for these antibodies were overlapping. Studies with GAD₆₅/GAD₆₇ chimeras indicated that the human monoclonals reacted with C-terminal epitopes. The human monoclonals, GAD6 and 3/5 mouse monoclonals inhibited serum autoantibody binding to ¹²⁵I-labelled GAD₆₅. Overall, the human monoclonals were of high affinity, reacted with C-terminal epitopes and showed evidence of antigen driven maturation; they represented only a proportion of the repertoire of autoantibodies to GAD₆₅ in the donor's serum and in the sera of patients with type-1 diabetes.     

用RIA法研究血循环中PHSA和AAA的存在情况

作者采用敏感的RIA法,分别检测179例人血清中PHSA和85例血清中的AAA。结果表明,血循环中的确存在PHSA,阳性检出率为11.7％,HBsAg阳性组和阴性组有显著性差异。AAA在血清中也有不同程度检出。作者根据检测情况,对弄清血循环中是否存在PHSA的意义,以及与Thung等提出的在HBV上的PHSA受体与血循环中PHSA结合再嗜肝的假说之关联等进行了讨论,并指出了用McAb在RIA检测方法中应注意的问题。     

Natural IgM: Beneficial Autoantibodies for the Control of Inflammatory and Autoimmune Disease

Natural IgM are highly represented in the circulation at birth, and these often autoreactive antibodies have been postulated to have innate-like properties and play crucial roles in apoptotic cell clearance, tissue homeostasis, and immune modulation. This review summarizes the known properties of these IgM autoantibodies, and the evidence that these anti-apoptotic cell IgM natural antibodies can regulate inflammatory responses through ancient pathways of the innate immune system that first arose long before the initial emergence of the adaptive immune system. While the regulatory contributions of these natural IgM autoantibodies are certainly not an essential and fundamental component of host defenses, these provide an additional layer to further protect the host. More importantly, these IgM antibody responses are highly inducible and their up-regulation can be a powerful means for the host to survive in a setting of chronic inflammation. The observed beneficial clinical associations for cardiovascular disease and autoimmunity, as well as opportunities for potential therapeutic implications are discussed.     

正常人血清中IgG类抗角蛋白自身抗体的免疫印迹分析

抗角蛋白自身抗体是正常人天然自身抗体的一部分。本研究用一组角蛋白对正常人血清中的ＩｇＧ 类抗角蛋白抗体进行了免疫印迹分析。２１ 份正常人血清均可识别至少一种角蛋白成分， 可区分出１４ 种反应性模式。３ 份合并血清的反应性模式高度均一。结果表明， 血清抗角蛋白抗体普遍存在， 但在人群中呈高度异质性； 不同个体血清可互补。角蛋白 抗角蛋白系统可能是天然自身抗体研究的一个理想模式系统。     

Inhibition of PDC-E2 Human Combinatorial Autoantibodies by Peptide Mimotopes

Immunohistochemical studies have shown that a unique immunoreactive molecule is present near the apical region of human biliary epithelial (BE) cells in patients with primary biliary cirrhosis (PBC). This can be visualized by confocal microscopy in PBC livers using a number of unique monoclonal antibodies to the E2 component of pyruvate dehydrogenase complex (PDC-E2), the autoantigen most commonly recognized by antimitochondrial antibodies (AMA). One such antibody, the murine mAb C355.1 was used to identify peptide mimotopes of PDC-E2 by screening a random dodecapeptide phage library ON 159.2 to identify the possible biochemical nature of this apical staining molecule. Out of 36 independent clones, 29 showed a common sequence and seven other sequences were singly represented. Three common amino acid motifs (SYP, TYVS and VRH) were found among these eight sequences. Similar to C355.1, the human combinatorial antibodies derived from a patient with PBC, SP1 and SP4, recognize the inner lipoyl domain of PDC-E2. However, when these antibodies are used to stain PBC BE cells, SP4 stains the apical region of PBC BE cells with high intensity whereas SP1 produces only cytoplasmic staining. Competitive inhibition of immunohistochemical staining using PDC-E2 specific human combinatorial antibodies SP1 and SP4 was performed using five of the above dodecapeptides. Interestingly, the peptides selected with C355.1 differentially inhibited the binding of SP1 and SP4 to PBC BE cells. Finally, rabbit sera raised against one such peptide (WMSYPDRTLRTS) stained BE cells from patients with PBC with a higher intensity than controls. Comparable data was obtained with immunoelectronmicroscopy. These data suggest that a molecular mimic of PDC-E2 is present at the external aspect of PBC BE cells.     

Human Tonsil-Derived Follicular Dendritic-Like Cells are Refractory to Human Prion Infection in Vitro and Traffic Disease-Associated Prion Protein to Lysosomes

The molecular mechanisms involved in human cellular susceptibility to prion infection remain poorly defined. This is due, in part, to the absence of any well characterized and relevant cultured human cells susceptible to infection with human prions, such as those involved in Creutzfeldt-Jakob disease. In variant Creutzfeldt-Jakob disease, prion replication is thought to occur first in the lymphoreticular system and then spread into the brain. We have, therefore, examined the susceptibility of a human tonsil-derived follicular dendritic cell-like cell line (HK) to prion infection. HK cells were found to display a readily detectable, time-dependent increase in cell-associated abnormal prion protein (PrP^TSE) when exposed to medium spiked with Creutzfeldt-Jakob disease brain homogenate, resulting in a coarse granular perinuclear PrP^TSE staining pattern. Despite their high level of cellular prion protein expression, HK cells failed to support infection, as judged by longer term maintenance of PrP^TSE accumulation. Colocalization studies revealed that exposure of HK cells to brain homogenate resulted in increased numbers of detectable lysosomes and that these structures immunostained intensely for PrP^TSE after exposure to Creutzfeldt-Jakob disease brain homogenate. Our data suggest that human follicular dendritic-like cells and perhaps other human cell types are able to avoid prion infection by efficient lysosomal degradation of PrP^TSE.Variant Creutzfeldt-Jakob disease (vCJD) is a fatal neurodegenerative disease resulting from oral infection with the bovine spongiform encephalopathy (BSE) agent.¹ BSE and vCJD belong to a group of transmissible spongiform encephalopathies (TSE) in which the infectious agent or prion is believed to be a conformationally altered and aggregated form prion protein TSE (PrP^TSE) of the host-encoded cellular prion protein (PrP^C), replicated by a templated conformational conversion process that resembles seeded aggregation.² vCJD involves the lymphoreticular system, probably before neuroinvasion and the subsequent appearance of neurological symptoms.^3,4 Accumulation of PrP^TSE in the follicular dendritic cells (FDCs) residing in the germinal centers in the tonsil, spleen, appendix, and lymph node is a consistent feature of vCJD pathology.^5,6 Exposure of the United Kingdom population to BSE is thought to have been widespread and yet the resultant vCJD epidemic has thus far been limited to 177 cases [National CJD Research & Surveillance Unit (NCJDRSU), http://www.cjd.ed.ac.uk/documents/figs.pdf, last accessed August 30, 2013]. All clinical cases of definite vCJD that have been tested are of the prion protein gene PRNP codon 129 MM genotype (NCJDRSU, http://www.cjd.ed.ac.uk/documents/report20.pdf, last accessed August 30, 2013). However, studies based on PrP^TSE immunostaining of routine appendectomy, tonsillectomy, and autopsy tissues indicate that the prevalence of infection in the United Kingdom is higher than the incidence of clinical vCJD would seem to suggest and indicates that all three possible PRNP codon 129 genotypes (MM, MV, VV) are susceptible to infection with the vCJD/BSE agent.^7–11 [Summary results of the second national survey of abnormal prion prevalence in archived appendix specimens are available online (Health Protection Report, http://www.hpa.org.uk/hpr/archives/2012/news3212.htm#bnrmlprn, last accessed August 30, 2013)]. The mechanism by which cells interact with prions is only beginning to be defined.¹² Despite a wealth of data showing that cultured cells of a variety of species and phenotypes can propagate animal prions, evidence that cultured human cells have been infected with human prion agents is restricted to a single unconfirmed report from 1995. In this report, Ladogana et al¹³ described infection of the human neuroblastoma SH-SY5Y cell line (PRNP codon 129 genotype MM) with a brain homogenate from a sporadic CJD patient (also PRNP codon 129 genotype MM). However, the infection proved to be unstable¹³ and this initial success in infecting human cells with the CJD agent has not been capitalized on. Interestingly, RK13 rabbit kidney epithelial cells expressing human PrP were found to be resistant to prion infection when exposed directly to sporadic CJD prions.¹⁴Our previous investigations of human embryonic stem cells (hESC) showed extensive and rapid uptake and clearance of PrP^TSE present in crude homogenates of bovine and human prion disease brain tissue added directly to culture medium.¹⁴ Uptake and clearance in these hESC did not appear to depend on the species, form of CJD (sporadic or variant), or the PRNP codon 129 genotype of the inoculum or the hESC, suggesting involvement of general rather than specific uptake mechanisms.¹⁵ This is consistent with earlier observations made using rodent adapted scrapie prions and rodent cell lines.¹⁶ Considering the involvement of the lymphoreticular system in vCJD, we chose to investigate the mechanisms involved in human cellular susceptibility to prion infection and PrP^TSE intracellular fate using the human cell line HK, which is derived from human tonsillectomy tissue and shares features with FDCs.¹⁷ Our results demonstrate that the human FDC-like HK cells, as far as we can determine, are resistant to prion infection in vitro and that the endosome/lysosome compartments are the likely sites of PrP^TSE intracellular trafficking and degradation.