Lilia A. Retegui

The antigenic topography of human growth hormone

Murine monoclonal antibodies (MAb) have been used as tools to probe the antigenic topography of human growth hormone (hGH). Mapping experiments were carried out by testing the ability of paired MAb to bind simultaneously or separately to 125I-hGH. A putative three-dimensional model of the relative distribution of 20 hGH epitopes indicated that they covered the entire molecular surface, showing the following essential characteristics. A domain of unique hGH specificity representing approximately 20% of the whole area was detected, as well as the presence of a discontinuous band of immunological identity between hGH and human placental lactogen (hPL) occupying 30% of the molecular surface. The rest of the surface (about 50%) displayed only partial cross-reactivity with hPL. Three restricted antigenic areas were also recognized. One of them appeared to correlate with a conformational change induced by the adsorption of the protein to plastic surfaces and the other two showed cross-reactivity with human prolactin and heterologous GH, respectively.

Allosteric effects of monoclonal antibodies on human growth hormone.

We have previously shown that a monoclonal antibody (MAb) recognizing the human growth hormone (hGH) antigenic domain left exposed after binding to lactogenic receptors enhanced hGH binding probably through allosteric effects on the hormone binding site. Since receptors displaying different specificities would not recognize exactly the same hGH region, we explored whether some of our MAb could affect hGH binding to somatogenic receptors from rabbit liver and to human liver hGH-specific receptors.The effect of MAbAE5, AC8 and F11 on hGH binding was measured by determining the formation of125I-MAb:hGH:receptor complexes using two different experimental approaches. Results from procedure A, which involved the previous binding of the hormone to microsomes before adding125I-MAb, indicated that the hGH domain defined by epitopes AE5, AC8 and F11 is uncovered in the various hormone:receptor complexes.Procedure B was devised to reveal any alteration in the hGH molecule induced by the MAb. In this case preformed125I-MAb:hGH complexes were added to microsomes. Data showed that125I-MAb AE5:hGH complexes bound better to the various receptors than125I-MAb AE5 to hGH:receptor complexes. On the contrary, hGH previously bound to125I-MAb AC8 or125I-MAb F11 was less recognized by the receptors than the free hormone. Furthermore, binding of MAb AE5 or MAb F11 to hGH 20 K (a natural hGH variant lacking residues 32–46) also enhanced its affinity to the various receptors whereas MAb AC8 did not inhibit hGH 20 K binding.Results indicated that MAb recognizing the hGH antigenic area that remains unmasked after binding to different membrane-bound receptors are able to affect hormone binding site. MAb would induce either positive or negative allosteric changes in the hormone region involved in its binding to lactogenic, somatogenic and hGH-specific receptors.

The peptide specificities of the autoantibodies elicited by mouse hepatitis virus A59.

Abstract Synthetic decapeptides (N =206) covering the entire sequence of mouse liver fumarylacetoacetate hydrolase (FAH) were used to analyze the specificities of the autoantibodies (autoAb) elicited towards this enzyme in mice infected with mouse hepatitis virus (MHV). These autoAb bound mainly to N- and C-terminal FAH peptides, the most reactive sequences being 1–50 and 390–420, respectively. Surprisingly, although FAH sequence 1–50 shares a high degree of homology with various MHV proteins, the C-terminal portion does not. Moreover, whereas the autoAb reacted with homologous peptides surrounding residues 70, 160 and 360, non-similar sequences around residues 130, 210, 240, 250, and 300 were also recognized, indicating that autoAb were not restricted to epitopes with sequence homologies. There was also a lack of correlation between the amount of anti-MHV or anti-FAH antibodies produced and the reactivity towards the peptides. Moreover, the spectrum of peptides recognized by the autoAb of a given mouse did not change significantly with time, which suggests that the MHV-elicited autoimmune response does not induce an epitope recognition spreading. Finally, anti-FAH Ab produced after immunization with rat liver FAH recognized essentially the same mouse FAH regions than autoAb from MHV-infected mice. Results indicated that the induction of the autoAb is not only related to molecular or structural mimicry, but rather supports the Danger model, in which any aggression, in this case the MHV infection, is susceptible to trigger the production of autoAb.

Autoimmune hepatitis-like disease in C57BL/6 mice infected with mouse hepatitis virus A59

Abstract Mouse hepatitis virus A59 (MHV A59) induces autoantibodies (autoAb) to fumarylacetoacetate hydrolase (FAH), a soluble cytosolic enzyme present in the liver and kidneys, in various mouse strains. The aim of this work was to amplify and diversify the autoimmune response restricted to FAH through the use of the exogenous adjuvant called PADRE. Accordingly, C57BL/6 mice were chosen, because these animals respond to PADRE better than other mouse strains. Results presented herein indicate that, surprisingly, C57BL/6 mice developed signs of autoimmune hepatitis-like disease (AIH), including transient hypergammaglobulinemia, elevated transaminases, autoAb directed against different liver proteins and hepatic cellular infiltrates, indicating that a new model of experimental AIH could be generated by a viral inoculation. Furthermore, PADRE administration amplified the MHV effect, extending the duration of hypergammaglobulinemia and increasing the binding of autoAb as well as the degree of hepatic infiltrates. However, the adjuvant did not expand the time of the symptoms. Additionally, since plasmatic uric acid and high-mobility group box protein 1 (HGMB1) concentrations augmented in MHV- and/or PADRE-treated mice, it is suggested that both alarmins were probably involved in the spreading of the immune response induced by the viral infection and the adjuvant administration.

Identification of two liver proteins recognized by autoantibodies elicited in mice infected with mouse hepatitis virus A59

Western blot experiments showed that sera from mice infected with the mouse hepatitis virus strain A59 (MHV‐A59) contained autoantibodies (autoAb) that bound to a 40‐kDa protein present in liver and kidney extracts. No reaction was observed with extracts of the heart, muscles, spleen, brain and lung. The Ab cross‐reacted with a 40‐kDa protein from human, rat and sheep liver, but not withliver extracts from the silver side fish (Odontesthes bonariensis). No correlation was found between the development of the hypergammaglobulinemia that followed the viral infection and theoccurrence of the autoAb. Reactive immunoglobulins pertained to the IgG1, IgG2a and IgG2b subclasses, recognized cryptic epitopes and were detected from 10 days up to 8 weeks after MHV‐infection. The 40‐kDa protein was purified from mouse liver extracts by ion‐exchange chromatography, gel filtration and SDS‐PAGE. Because the N‐terminal was blocked, we digested the protein in‐gel with trypsin and sequenced various peptides. Results indicated a 100% homology of sequence between the protein recognized by the autoAb and liver fumarylacetoacetate hydrolase (FAH), the enzyme that mediates the last step of tyrosine catabolism. Additionally, a second protein recognized by the autoAb was detected during FAH purification steps and was identified as liver alcohol dehydrogenase.

Monoclonal antibodies to human growth hormone modulate its biological properties

Previous results indicated that monoclonal antibodies (mAbs) termed mAb AE5, mAb AC8 and mAb F11, recognizing the human growth hormone (hGH) region left exposed after binding to lactogenic, somatogenic and hGH-specific receptors, produce allosteric changes in the hormone which modify its binding properties. To study whether these mAbs could also influence hGH biological activity, experiments were carried out with Nb2 cells, a rat lymphoma cell line which proliferates in the presence of lactogenic hormones. Experiments involving previous binding of the hormone to receptors before adding 125I-mAbs indicated that the hGH domain defined by overlapped epitopes AE5, AC8 and F11 is uncovered in hGH when it is bound to the cell membranes. To reveal any alteration in the hGH molecule induced by the mAbs, preformed 125I-mAb:hGH complexes were added to the cell membranes. Data showed that 125I-mAb AE5:hGH complexes bound better to the receptors than free hormone. On the contrary, hGH previously bound to 125I-mAb AC8 or 125I-mAb F11 was poorly recognized by Nb2 receptors. Furthermore, both mAbs AC8 and F11 strongly inhibited 125I-hGH binding to Nb2 cell membranes and hGH-induced Nb2 cell proliferation whereas mAb AE5 enhanced both hormone binding and hGH mitogenic effect. Additionally, since mAb AC8 is directed towards an epitope shared by hGH and human placental lactogen (hPL), it was also shown that this mAb could impair hPL biological activity even though it recognizes the hPL region left exposed in hPL:Nb2 cell receptor complexes. Data presented in this work suggest that mAbs directed to the hGH or hPL regions unmasked after binding to Nb2 cell receptors produce allosteric alterations in the binding properties of these hormones leading to either enhancement or decrease of their biological activities.

A monoclonal antibody recognizing an epitope shared by receptors for growth hormone, prolactin, interleukin 2 and interleukin 6

Monoclonal antibody (MAb) termed R7B4 was generated throughout the idiotypic-antiidiotypic network from mice immunized with human and bovine growth hormones (GH). The Ab was selected on the basis that it did not recognize human GH (hGH) neither insolubilized nor in solution but inhibited 125I-hGH binding to receptors from rat and rabbit liver and from Nb2-cell membranes.Since it inhibited Nb2-cell mitogenesis stimulated by hGH, prolactins or placental lactogens, MAb R7B4 behaved as an antagonist of lactogenic hormones. Furthermore, the Ab impaired proliferative activity of interleukin 2 (IL-2) on Nb2 cells as well as growth of 7TD1 cells, an interleukin 6 (IL-6) dependent hybridoma not expressing GH receptors.Biotin-labeled MAb R7B4 specifically bound to rat liver microsomes, and the Ab was able to recognize Nb2 and 7TD1-cell membranes as shown by flow cytometry experiments. However, MAb binding was not hampered by hGH, indicating that the Ab did not mimic GH binding site to receptors. Immunoblot assays indicated that rat and rabbit liver as well as Nb2-cells membrane antigens recognized by MAb R7B4 were similar to those revealed by a MAb directed to prolactin receptors. In addition, MAb R7B4 was able to detect two bands probably corresponding to the somatogenic receptor in rabbit liver microsomes as well as three different proteins in 7TD1-cells showing molecular weights similar to those of the IL-6 receptor complex.Results suggest that MAb R7B4 is directed to an epitope shared by receptors for lactogenic and somatogenic hormones, IL-2 and IL-6. To our knowledge, these data are the first experimental evidence of the existence of structural similarity between some of the receptors grouped in the cytokine receptor superfamily.

Synergistic monoclonal antibodies‘ interactions and their use for determination of antibody specificities

Three monoclonal antibodies (MAb 3C11, F11 and 10D6) to human growth hormone (hGH) recognize independent epitopes and show mutually enhancing properties. Thus, 125I-hGH binding to each of these MAb augmented significantly in the presence of each one of the other two MAb. Moreover, preincubation of the hormone with paired MAb gave rise to ternary complexes (Ag:Ab1:Ab2) which bound better than the free tracer to the third MAb previously captured on a solid-phase. Highly stable quaternary complexes (Ag:Ab1:Ab2:Ab3) were thus formed. Since Fab fragments from the three MAb displayed the same behavior as the whole Ab molecule, neither the formation of multimolecular cyclic complexes nor the occurrence of interactions through Fc fragments could explain the reciprocal MAb binding enhancement. Therefore, the results obtained suggest that MAb 3C11, F11 and 10D6 produce some modification in the Ag, each one improving the binding of the two other MAb. Additionally, the inhibition of the formation of quaternary complexes between the MAb and hGH was used to evaluate specific Ab populations in polyclonal antisera, avoiding the masking effect of enhancing Ab. The results obtained indicate that Ab directed to the hGH antigenic domains defined by MAb 3C11, F11 and 10D6 could be detected in spite of the presence of enhancing Ab to all three MAb.

Sequence similarity and structural homologies are involved in the autoimmune response elicited by mouse hepatitis virus A59

Abstract The features of autoantibodies (autoAb) to liver fumarylacetoacetate hydrolase (FAH) elicited in mice infected with mouse hepatitis virus (MHV) were studied by ELISA and western-blot competition assays. All sera tested contained Ab to cryptic FAH epitopes according with results from western-blot tests, whereas ELISA data indicated that some of these same sera did recognize native epitopes of the autoantigen (autoAg). Such differences were detected in individual sera from various mouse strains, and were ascribed to the fact that proteins insolubilized on solid supports expose a variety of conformational and cryptic antigenic determinants. On the other hand, whereas results from both experimental protocols showed that anti-MHV Ab did not cross-react with the soluble autoAg, the opposite situation did not show analogous results. Thus, binding of autoAb to insolubilized FAH could be inhibited by MHV depending on the mouse serum or the experimental protocol used. Additionally, a set of synthetic homologous peptides from mouse FAH and various viral proteins was employed to analyze the Ab repertoire of MHV-infected mice. Results indicated that two homologous peptides were recognized by most Ab: the N-terminal sequences (1–10) from FAH and the nucleocapside, both sharing 50% of identity, and sequence 2317–2326 of the RNA polymerase, a peptide showing 30% of identity with FAH 11–20. Results indicated that MHV-infection triggers at least three distinct Ab populations: anti-MHV, anti-FAH and cross-reacting Ab. This cross-reaction implies either sequential or conformational epitopes from both the viral proteins and the autoAg and may differ between individuals.

Uric acid and HMGB1 are involved in the induction of autoantibodies elicited in mice infected with mouse hepatitis virus A59

We have shown that mice infected with mouse hepatitis virus A59 develop autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hydrolase (FAH). Because it has been proposed that the immune system is stimulated by alarm signals called damage-associated molecular patterns or alarmins, we investigated the participation of uric acid and high-mobility group box protein 1 (HMGB1) in the autoimmune response elicited by mouse hepatitis virus (MHV). Mice subjected to MHV infection had increased plasmatic uric acid concentration that significantly decreased after 20 days of daily treatment with allopurinol and, simultaneously, autoAb to FAH were undetected. Furthermore, this autoAb disappeared after 30 days of treatment with ethyl pyruvate, along with a substantial reduction in serum HMGB1 concentration. Both results indicated a remarkable relationship between the autoimmune process induced by the virus and uric acid and HMGB1 liberation. Unexpectedly, it was found that allopurinol and ethyl pyruvate inhibited the release of both uric acid and HMGB1. Because HMGB1 is activated through binding to interleukin 1β, and that this cytokine is produced by the NLRP3 inflammasome that could be stimulated by uric acid, we propose that both alarmins could be acting in concert with the induction of the autoAb to FAH in MHV-infected mice.