To further our studies on the functional significance of platelet-derived PPARγ, we needed antibodies that could detect PPARγ in blood and blood cells. In previous studies, we used the Biomol and Calbiochem anti-PPARγ PoAbs (Feldon et al., 2006
, Ray et al., 2008
, O'Brien et al., 2008
); however, per product specification sheets, neither of these PoAbs could be used to detect PPARγ in the presence of serum albumin. Also, for unknown reasons during the course of these studies, the Biomol and Calbiochem PoAbs became no longer available, prompting us to produce our own anti-PPARγ rabbit PoAbs and MoAbs. Unexpectedly, during the course of production and characterization of PoAbs and MoAbs against PPARγ peptide immunogens, our work produced two key findings. First, we observed significant and unusual pathology in the immunized rabbits and mice over the time course of immunization. Second, although the PoAbs produced were monospecific for PPARγ, the MoAbs produced against the same 14mer and 19mer peptide-immunogens crossreacted with PPARα and PPARβ/δ.
The remarkable pathology we observed in both rabbits and mice suggested that production of anti-PPARγ antibodies produced an immune complex disease similar to immune thrombocytopenia. In particular, we observed low platelet counts in rabbits immunized with the PPARγ 14mer peptide-immunogen. In addition, signs of increased vascular permeability were observed in all four immunized rabbits including unexplained purpura, enlarged ovaries likely due to edema, plasma leakage and excessive bleeding from ruptured and necrotic ovarian follicles. Excessive hemorrhaging was observed in the spleens of immunized mice and in the lymph nodes and intraperitoneal tumors of the mice injected with anti-PPARγ hybridoma cells for MoAb production in ascites fluid. Furthermore, platelet counts were substantially reduced in the anti-PPARγ-14mer 3C11 (IgM isotype) ascites mice, but not in the D73H ascites mice producing an IgM isotype MoAb against the Bβ-chain of human fibrinogen (Rybarczyk et al., 2000
). Platelet depletion and increased MPV reflect the state of thrombogenesis (Hekimsoy et al., 2004
). High MPV is associated with low platelet counts due to platelet destruction (immune thrombocytopenia) and platelet activation (Coban et al., 2008
, Sullivan et al., 1995
). Increase in MPV occurs in patients with metabolic syndrome, stroke and diabetes mellitus (Zuberi et al., 2008
), contributing to the increased vascular complications and endothelial cell dysfunction observed (Hekimsoy et al., 2004
). Although we observed increased MPV in only one rabbit with low platelet counts, platelet functions were clearly compromised in all four rabbits and 3C11 ascites mice. Together, these results support the hypothesis that immune-mediated destruction of platelets occurred in animals immunized with the PPARγ peptide-immunogens due to crossreactivity of the elicited anti-PPARγ antibodies with platelet-derived PPARγ.
The mechanisms by which anti-PPARγ antibodies induce sporadic or transient thrombocytopenia are presently unknown. However, immune-mediated destruction of platelets occurs during idiopathic/immune thrombocytopenia purpura, which is frequently caused by IgG isotype antibodies against platelet membrane glycoprotein complexes αIIb-IIIa or Ib-IX (Hou et al., 1995
). Furthermore, antibodies against GP1bα are frequently used to induce thrombocytopenia in mice. Acute thrombocytopenia occurs within 60 min after injecting mice with purified rat PoAb IgG raised against platelet GP1bα (R300) and, according to the R300 product specification sheet, mouse platelet counts can be reduced up to 95% using 4 μg/g body weight and remain low for 48 to 72 hours. Therefore, we determined whether passive administration of purified anti-PPARγ-14mer or anti-PPARγ-19mer IgG could reduce platelet counts in naïve mice using both acute and chronic models of experimental immune-mediated thrombocytopenia. Neither anti-PPARγ-14mer nor anti-PPARγ-19mer IgG induced thrombocytopenia in the acute or chronic model of immune-mediated platelet destruction. However, megakaryocytopoiesis was enhanced 4–8–fold in mouse spleens of mice treated with anti-PPARγ-14mer or anti-PPARγ-19mer IgG suggesting that compensatory platelet production maintained normal levels of blood platelets. Enhanced production of megakaryocytes in the bone marrow of all four rabbits immunized with PPARγ synthetic peptides was also observed. Compensatory platelet production due to enhanced megakaryocytopoiesis would explain the thrombocytopenia and bleeding anomalies observed in both rabbits and mice immunized with PPARγ peptide-immunogens. These data further support the hypothesis that immune-mediated destruction of platelets occurred in animals immunized with the PPARγ peptide-immunogens due to crossreactivity of the elicited anti-PPARγ antibodies with platelet-derived PPARγ.
The advent of MoAb production to design an antibody that exquisitely recognizes a defined epitope of a given molecule using the method developed by Kohler and Milstein in 1975 (Kohler and Milstein, 1975
) has revolutionized basic biomedical research, diagnostics and disease treatment. MoAbs produce only one specific antibody isotype against a defined epitope; however, there is no guarantee that this antibody is monospecific against the native antigen. Although the PoAbs we produced against the PPARγ-14mer and PPARγ-19mer peptide-immunogens were monospecific for rPPARγ, the MoAbs we produced against the same peptide-immunogens crossreacted with rPPARα and rPPARβ/δ in addition to rPPARγ. Indeed, two commercially available anti-PPARγ antibodies from Santa Cruz Biotechnology show strikingly similar results as reported herein. PoAb anti-PPARγ sc-7196 is monospecific for rPPARγ while MoAb anti-PPARγ sc-7273 crossreacts with rPPARα, rPPARβ/δ and rPPARγ. The peptide immunogen used to produce sc-7273 maps to the last 25 amino acids of PPARγ, an epitope structurally distinct from the 14mer and 19mer used in this study, suggesting that selection of the immunodominant hybridomas producing these anti-PPARγ MoAbs responded to a peptide conformation and primary structure shared by PPARα, PPARβ/δ and PPARγ. The lack of monospecificity of the anti-PPARγ MoAbs described in this report and available commercially (sc-7273) is a striking example of one of the major pitfalls associated with production of MoAbs against closely related molecules—monoclonicity does not guarantee monospecificity. Santa Cruz anti-PPARγ MoAb, sc-7273, is frequently reported in the literature for immunodetection of PPARγ. Currently, 132 citations using this antibody are listed on the Santa Cruz web page (http://www.scbt.com/datasheet-7273-ppargamma-e-8-antibody.html
). In that the apparent molecular weights of the three PPAR isoforms fall within 49–54 kDa, it is important that the specificities of antibodies used for detection are thoroughly vetted.
Amino acid stretches as small as 4–6 residues define highly immunogenic determinants (Hopp and Woods, 1981
), and the potential for representation of such determinants in unrelated molecules is high. The ability to produce highly specific MoAbs against peptide-immunogens is further complicated when the target of interest is a member of a closely related gene family such as the PPARs. The hybridoma cell line selected for a specific MoAb produces the antibody reactivity of one plasma cell that responded to the presentation of a single peptide-immunogen conformation in solution selected from an undetermined number of peptide-immunogen conformations presented to the immune system during production of PoAbs. Small peptide sequences inherently lack defined secondary and tertiary structure. Thus, the number of conformations that synthetic peptides adopt and the length of time such a conformation is maintained is unknown. Indeed, development of anti-peptide vaccines to prevent HIV infection has been hindered by the lack of structure in aqueous solution of the major neutralizing determinant on envelope protein gp120 of HIV-1 (Chandrasekhar et al., 1991
). Our strategy for selection of primary structure for choice of peptide immunogens takes advantage of known parameters that restrict random structure (Section 2.2). However, our strategy is not foolproof even though we successfully produced a number of anti-peptide MoAbs against closely related molecules (Simpson-Haidaris et al., 1989a
, Simpson-Haidaris et al., 1989b
, Fay et al., 1991
, Odrljin et al., 1996
, Meh et al., 2001
Others have shown the lack of monospecificity of MoAbs as well. For example, a MoAb that binds preferentially to integrin αIIbβ3 on activated platelets (7E3) crossreacts with integrin CD11b/CD18 (Mac-1) on activated monocytes (Simon et al., 1997
, Schwarz et al., 2002
). MoAb 7E3, the parent antibody of abciximab (ReoPro), is used to prevent platelet aggregation in patients undergoing angioplasty (EPIC-Investigation, 1994
). It is thought that inhibition of CD11b/CD18-dependent adhesion and αIIb/β3-dependent function may jointly contribute to the regulation of vascular repair and sustained clinical benefits observed with abciximab after angioplasty (Simon et al., 1997
). In this example, crossreactivity of MoAb 7E3 with integrin CD11b/CD18 on monocytes appears to have a beneficial clinical effect; however, such a positive result may not always hold true. Additionally, there are the seven publications regarding the lack of specificity of multiple antibodies from commercial and academic sources that are used for mapping receptors of therapeutic interest, including antibodies specific for adrenergic, muscarinic, and dopaminergic receptors (Kirkpatrick, 2009
). Because it is important to properly identify the appropriate PPAR isoform in various tissues, clarity in the reactivity of MoAbs and PoAbs against the PPAR isoforms (α, β/δ and γ), which exhibit highly conserved primary structure, functional domains and nearly identical apparent molecular weights, is essential.
PPARγ agonists, including the thiazolidinediones, exert beneficial effects on glycemic control and reduction of inflammatory cardiovascular risk factors, making them attractive agents for treatment of type 2 diabetics at high risk for cardiovascular disease (Spinelli et al., 2008
). PPARs exert anti-inflammatory effects by inhibiting the induction of pro-inflammatory cytokines, adhesion molecules and extracellular matrix proteins or by stimulating the production of anti-inflammatory molecules (Kostadinova et al., 2005
). Moreover, PPARγ agonists inhibit platelet aggregation and release of storage granule constituents such as CD40 ligand and thromboxane A2
(Akbiyik et al., 2004
), potent mediators of inflammation and cardiovascular disease. Systemic inflammation is clearly linked with adverse prognosis in patients with cancer as well (Sahni et al., 2009
). Release of platelet granule constituents plays a critical role in maintaining the integrity of tumor vasculature to prevent hemorrhaging, and neither platelet aggregation nor thrombi formation is required for this function (Ho-Tin-Noe et al., 2008
, Ho-Tin-Noe et al., 2009
). It is intriguing to speculate that release of platelet-derived PPARγ and uptake by vascular endothelium, perhaps from microparticles, plays a role in maintaining the integrity of tumor vasculature to prevent bleeding. In that PPARγ ligands promote apoptosis, induce cell-cycle arrest, promote cell differentiation and inhibit tumor angiogenesis and metastasis, PPARγ is considered a potential therapeutic target in hematological malignancies (Garcia-Bates et al., 2008a
, Garcia-Bates et al., 2008b
) and cancers of epithelial origin (Panigrahy et al., 2005
, Tachibana et al., 2008
, Murphy and Holder, 2000
). Furthermore, PPARγ ligands decrease vascular permeability and leukostasis associated with diabetic retinopathy (Yanagi, 2008
), suggesting that PPARγ plays a global role in maintenance of vascular homeostasis.
In summary, the observations reported herein support the assertion that the validity of MoAbs and PoAbs produced by academic investigators and commercial vendors for biomedical research and ultimately clinical therapy cannot be taken at face value. A thorough vetting of the specificity of such reagents is required to allow accurate interpretation of experimental data or clinical trial outcomes proving the identity or efficacy of putative drug targets.