Development and characterization of murine monoclonal antibodies to the latency-associated peptide of transforming growth factor beta 1.

Transforming growth factor beta (TGF-beta) is a multifunctional peptide that controls proliferation, differentiation, and other functions in a variety of cell types. Transforming growth factor beta activities have been implicated in a variety of diseased states including arthritis, prostate cancer, and AIDS, and in the repair of tissue injury caused by trauma, burns, and surgery. We describe the development and characterization of novel murine monoclonal antibodies (MAbs) to the latency-associated peptide (LAP) of TGF-beta 1, and the subsequent development of an ELISA for the detection and quantitation of TGF-beta 1-LAP in buffer and serum matrices. Fusion of immune splenocytes with myeloma cells yielded 576 hybridomas, 110 of which were antibody secreting. Five were selected for extensive characterization. Clinically, the MAbs described here should be valuable for studying potentially abnormal production and/or function of the LAP, and its relationship to TGF-beta.

Pharmacokinetics and tissue distribution of recombinant human transforming growth factor beta 1 after topical and intravenous administration in male rats.

Recombinant human transforming growth factor beta (rhTGF-beta 1) enhances the healing process after topical application to various animal wound models. A detailed pharmacokinetic and tissue distribution study was performed to support the clinical development of rhTGF-beta 1 for wound healing indications. Rats received radioiodinated or unlabeled rhTGF-beta 1 as an intravenous (iv) bolus or as a topical formulation applied to a full thickness wound. Plasma concentrations of TGF-beta 1 were estimated from TCA-precipitable radioactivity or were measured by ELISA. Following iv administration, the initial half-life was rapid (< 11 min), regardless of whether radiolabeled or unlabeled rhTGF-beta 1 was used. The terminal half-life was long (163 min) when the test material was radioiodinated and administered as a trace dose and relatively short (< or = 61 min) when given at high doses and assayed by ELISA. Analysis of plasma radioactivity by SDS-PAGE revealed a time-dependent clearance of the 25-kDa parent molecule without a significant appearance of lower molecular weight radiolabeled metabolites. The majority of the radioactivity was associated with highly perfused organs, known iodide elimination pathways, and the thyroid at 1 and 8 hr after iv injection. After topical administration of a high dose (0.8 mg/kg), no immunoreactive TGF-beta 1 was detectable in plasma samples taken over a 48-hr period. However, trace amounts (< or = 0.05 ng/mL) of acid-precipitable radioactivity were detected in plasma after topical application of [125I]rhTGF-beta 1 (1 microgram/kg, 126 microCi/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta primes macrophages to express inflammatory gene products in response to particulate stimuli by an autocrine/paracrine mechanism.

Macrophages maintain an essential role in orchestrating the host inflammatory response by selectively mobilizing portions of their large secretory repertoire in response to phagocytic as well as other stimuli. For example, after exposure to the inflammatory particulate stimulus zymosan (or its derivative beta 1,3-glucan), monocyte/macrophages synthesize and release lysosomal hydrolases, mobilize arachadonic acid, and secrete cytokines such as TNF-alpha and IL-8. However, the mechanisms by which particulate stimuli promote the selective synthesis and release of macrophage-derived inflammatory gene products are unknown. Given the previously reported potential of transforming growth factor-beta (TGF-beta) as an important mediator of the inflammatory response in vivo, we investigated the role of TGF-beta in the regulation of particulate-induced macrophage inflammatory gene expression. We determined that TGF-beta primed macrophages to synthesize lysosomal hydrolases and express platelet-derived growth factor-B mRNA transcripts in response to both submaximal doses of beta 1,3-glucan and the nonspecific phagocytic stimulus latex particles, which by themselves did not induce expression of either inflammatory gene product. The endogenous production of active TGF-beta was shown to regulate inflammatory gene expression by demonstrating that: 1) beta 1,3-glucan stimulated both TGF-beta mRNA expression and protein release into conditioned media; 2) supernatants from stimulated macrophages primed for lysosomal hydrolase synthesis, and this effect was blocked by anti-TGF-beta antibodies; and 3) anti-TGF-antibodies blocked beta 1,3-glucan-stimulated lysosomal hydrolase synthesis. Collectively, these data describe a novel function for TGF-beta as a priming agent for macrophage inflammatory gene expression and suggest a mechanism for local amplification of the inflammatory response.

An evaluation of mechanisms by which tolerance to organ-specific antigens is lost using a transgenic mouse model.

Patients with type I diabetes lose immunologic tolerance to beta cell Ag and produce anti-self (beta cell) antibodies. We have examined mechanisms by which this self tolerance is lost using transgenic mice in which islet cells express human CD4. These mice are immunologically tolerant to this human protein; the tolerance appears to be characterized by the presence of self reactive B cells and nonresponsive T cells. Autoantibodies can be induced by immunization with CD4 in CFA; this autoantibody response is accompanied by minor peri-islet inflammation but diabetes is not induced. Tolerance can also be broken by provision of an independent covalently linked T cell epitope. Although this mimic can lead to the appearance of an autoantibody response, the autoimmune response can not be maintained by the endogenous Ag. We have also demonstrated that an inflammatory lesion leading to death of the CD4-containing beta cells may not be sufficient to break tolerance. These results have implications for potential mechanisms relating to the appearance of autoantibodies in patients with type I diabetes.

The measurement of staphylococcal protein A by ELISA in immunoglobulin preparations.

Chicken antibodies were used to develop an ELISA for the quantitation of parts-per-million levels of protein A in the purification of immunoglobulins or immunoglobulin-like molecules. Quantitation of protein A in the presence of excess human or murine immunoglobulins in this assay was compared with that obtained in ELISAs developed with rabbit antibodies specific either to protein A or to other molecules. Experiments demonstrate that protein A is bound to the immunoglobulins being purified and that this binding affects subsequent recognition by the antibodies used for the assay. Because of these effects and because fragments of protein A might not be detected in assays which rely on Fc binding of protein A, chicken antibodies that bind protein A specifically are an advantage for the quantitation of this protein by ELISA. In addition, comparison of the effect of different types of immunoglobulins on the protein A standard curve suggests that alternatives to including the immunoglobulin under purification with the standards can be utilized.

The autocrine production of transforming growth factor-beta 1 during lymphocyte activation. A study with a monoclonal antibody-based ELISA.

We describe the production and characterization of three mAb to transforming growth factor-beta (TGF-beta) and the use of two of them for the development of a TGF-beta 1-specific ELISA and for the study of the regulation of immune function in vitro. All three mAb bound recombinant human TGF-beta 1 (rHuTGF-beta 1) with high affinity and recognized the dimer form of this molecule in immunoblots. mAb 2G7 immunoprecipitated rHuTGF-beta 1, TGF-beta 2, and rHuTGF-beta 3 and neutralized the growth inhibitory activity of all three molecules in vitro on mink lung epithelial-like cells, Mv1Lu, indicating a shared neutralization epitope. mAb 4A11 neutralized and immunoprecipitated only rHuTGF-beta 1, and mAb 12H5 immunoprecipitated rHuTGF-beta 1 but had no effect on the bioactivity of either rHuTGF-beta 1, TGF-beta 2, or rHuTGF-beta 3. These results suggest that a second neutralization epitope may be unique to TGF-beta 1. The ELISA was developed with mAb 4A11 and 12H5, with a range of 0.63 to 40 ng/ml, i.e., a sensitivity of 0.63 ng/ml or 63 pg/sample. The assay is accurate, precise, and specific for the active but not the inactive or latent TGF-beta 1 complex and fails to react with TGF-beta 2, rHuTGF-beta 3, inhibin A, and activin A. Supernatants obtained from serum-free cultures of human PBMC from multiple donors contained significant quantities of TGF-beta 1 (3 to 15 ng/ml), which was detected in the ELISA only after pH 2 treatment to convert latent TGF-beta to the active form. Treatment of the PBMC with either recombinant human IL-2 (rHuIL-2) or PHA-P/PMA enhanced the production of latent TGF-beta 1. mAb 4A11 and 2G7, but not mAb 12H5 enhanced both the proliferative response of PBMC to rHuIL-2/rHuTNF-alpha and PHA-P and the development of the rHuIL-2/rHuTNF-alpha treated PBMC into LAK cells with cytotoxic activity against COLO target cells. These findings suggest that although PBMC secrete latent TGF-beta 1, mechanisms that convert the latent TGF-beta complex into an active form exist in vitro and that the endogenously produced TGF-beta can regulate immune functions in an autocrine fashion.

Cyclic AMP response to recombinant human relaxin by cultured human endometrial cells--a specific and high throughput in vitro bioassay.

A specific and high throughput 96-well format bioassay for recombinant human relaxin (rhRLX) has been developed using human endometrial cells (NHE cells). rhRLX caused a time- and dose-dependent stimulation of cyclic AMP (cAMP) with 1/2 maximal activity of 3.56 +/- 0.65 ng/ml (n = 30). The range of the standard curve was 0.39 to 25 ng/ml with interplate precision of 17 and 22% CV for high and low controls respectively. The cAMP response requires forskolin and 3-isobutyl-1-methylxanthine, and is enhanced by prostaglandin E2 and F2 alpha. The NHE cells do not respond to A or B chains of rhRLX, or a whole array of hormones. Preincubation of rhRLX with specific monoclonal antibody completely abolished the cAMP response. This bioassay has been used to determine the biological activity of several manufactured lots of recombinant human relaxin.

An enzyme-linked immunosorbent assay to study human relaxin in human pregnancy and in pregnant rhesus monkeys.

A sensitive and specific double-antibody enzyme-linked immunoassay, using a synthetic analogue of human relaxin for standard and immunogen, was developed for the measurement of human relaxin (hRLX) in serum and plasma. No cross-reactivity was observed for human insulin, human insulin-like growth factor-I, hGH, human chorionic gonadotropin, hFSH, hLH or human prolactin. The assay was used to monitor RLX concentrations in samples from men, non-pregnant and pregnant women, and in pregnant rhesus monkeys infused with hRLX. RLX was not detected in serum from men nor from non-pregnant women, while a concentration of 600 ng/l was measured in pooled sera from two pregnant women (pregnancies achieved by in-vitro fertilization). Immunoreactive RLX (1.1 micrograms/g) was found in human corpora lutea taken from ectopic pregnancies at 7 weeks. In an experiment with a pregnant rhesus monkey infused with human RLX analogue, less than 1.5% of the maternal concentration was measured in the fetal circulation. Even though preliminary, these data suggest a low level of transfer of human analogue relaxin across the placenta in a rhesus monkey. Further studies of the physiology of RLX in human pregnancy will be facilitated by the availability of this immunoassay.