Growth and the Growth Hormone-Insulin Like Growth Factor 1 Axis in Children With Chronic Inflammation: Current Evidence, Gaps in Knowledge, and Future Directions.

Growth failure is frequently encountered in children with chronic inflammatory conditions like juvenile idiopathic arthritis, inflammatory bowel disease, and cystic fibrosis. Delayed puberty and attenuated pubertal growth spurt are often seen during adolescence. The underlying inflammatory state mediated by proinflammatory cytokines, prolonged use of glucocorticoid, and suboptimal nutrition contribute to growth failure and pubertal abnormalities. These factors can impair growth by their effects on the GH-IGF axis and also directly at the level of the growth plate via alterations in chondrogenesis and local growth factor signaling. Recent studies on the impact of cytokines and glucocorticoid on the growth plate further advanced our understanding of growth failure in chronic disease and provided a biological rationale of growth promotion. Targeting cytokines using biological therapy may lead to improvement of growth in some of these children, but approximately one-third continue to grow slowly. There is increasing evidence that the use of relatively high-dose recombinant human GH may lead to partial catch-up growth in chronic inflammatory conditions, although long-term follow-up data are currently limited. In this review, we comprehensively review the growth abnormalities in children with juvenile idiopathic arthritis, inflammatory bowel disease, and cystic fibrosis, systemic abnormalities of the GH-IGF axis, and growth plate perturbations. We also systematically reviewed all the current published studies of recombinant human GH in these conditions and discussed the role of recombinant human IGF-1.

Inhibition of IGF-I-related intracellular signaling pathways by proinflammatory cytokines in growth plate chondrocytes.

BACKGROUND: Children with chronic inflammatory diseases suffer from severe growth failure associated with resistance toward the anabolic action of insulin-like growth factor I (IGF-I). We hypothesized that proinflammatory cytokines interfere with IGF-I signaling. METHODS: We used the mesenchymal chondrogenic cell line RCJ3.1C5.18 as a model of the growth plate. Cell proliferation was assessed by [(3)H]-thymidine-uptake and differentiation by gene expression (quantitative reverse-transcriptase PCR) of specific differentiation markers. Key signaling molecules of the respective IGF-I-related intracellular pathways were determined by western immunoblotting. RESULTS: Coincubation of the proinflammatory cytokines interleukin (IL)-1ß (10 ng/ml), IL-6 (100 ng/ml), or tumor necrosis factor-α (50 ng/ml) with IGF-I inhibited IGF-I-driven cell proliferation by 50%, while baseline cell proliferation was not altered. These cytokines attenuated the IGF-I-induced phosphorylation of AKT as a key signaling molecule of the phosphatidylinositol-3 kinase pathway by 30-50% and the phosphorylation of ERK as a key signaling molecule of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by 50-75%. Also, IGF-I-enhanced chondrocyte differentiation was inhibited by these proinflammatory cytokines. CONCLUSION: The insensitivity toward the anabolic action of IGF-I in the growth plate in conditions of chronic inflammation is partially due to inhibition of IGF-I-specific signaling pathways by proinflammatory cytokines, which affect both IGF-I-driven chondrocyte proliferation and differentiation.

Roles of neutrophil-mediated inflammatory response in the bony repair of injured growth plate cartilage in young rats.

Injured growth plate cartilage is often repaired by bony tissue, resulting in impaired bone growth in children. Previously, injury-induced, initial inflammatory response was shown to be an acute inflammatory event containing predominantly neutrophils. To examine potential roles of neutrophils in the bony repair, a neutrophil-neutralizing antiserum or control normal serum was administered systemically in rats with growth plate injury. The inflammatory response was found temporally associated with increased expression of neutrophil chemotactic chemokine cytokine-induced neutrophil chemoattractant-1 and cytokines TNF-alpha and IL-1beta. Following the inflammatory response, mesenchymal infiltration, chondrogenic and osteogenic responses, and bony repair were observed at the injury site. Neutrophil reduction did not significantly affect infiltration of other inflammatory cells and expression of TNF-alpha and IL-1beta and growth factors, platelet-derived growth factor-B and TGF-beta1, at the injured growth plate on Day 1 and had no effects on mesenchymal infiltration on Day 4. By Day 10, however, there was a significant reduction in proportion of mesenchymal repair tissue but an increase (although statistically insignificant) in bony trabeculae and a decrease in cartilaginous tissue within the injury site. Consistently, in antiserum-treated rats, there was an increase in expression of osteoblastic differentiation transcription factor cbf-alpha1 and bone matrix protein osteocalcin and a decrease in chondrogenic transcription factor Sox-9 and cartilage matrix collagen-II in the injured growth plate. These results suggest that injury-induced, neutrophil-mediated inflammatory response appears to suppress mesenchymal cell osteoblastic differentiation but enhance chondrogenic differentiation, and thus, it may be involved in regulating downstream chondrogenic and osteogenic events for growth plate bony repair.

Matrix metalloproteinases are not essential for aggrecan turnover during normal skeletal growth and development.

The growth plate is a transitional region of cartilage and highly diversified chondrocytes that controls long bone formation. The composition of growth plate cartilage changes markedly from the epiphysis to the metaphysis, notably with the loss of type II collagen, concomitant with an increase in MMP-13; type X collagen; and the C-propeptide of type II collagen. In contrast, the fate of aggrecan in the growth plate is not clear: there is biosynthesis and loss of aggrecan from hypertrophic cartilage, but the mechanism of loss is unknown. All matrix metalloproteinases (MMPs) cleave aggrecan between amino acids N341 and F342 in the proteinase-sensitive interglobular domain (IGD), and MMPs in the growth plate are thought to have a role in aggrecanolysis. We have generated mice with aggrecan resistant to proteolysis by MMPs in the IGD and found that the mice develop normally with no skeletal deformities. The mutant mice do not accumulate aggrecan, and there is no significant compensatory proteolysis occurring at alternate sites in the IGD. Our studies reveal that MMP cleavage in this key region is not a predominant mechanism for removing aggrecan from growth plate cartilage.

A monoclonal antibody distinguishes growth cartilage from other types of cartilage: a new probe for osteogenic cartilage.

Monoclonal antibodies (mAbs) were raised by injection of a homogenate of cultured growth cartilage (GC) cells from young rabbit ribs. These mAbs were examined by immunohistochemical staining for their reactivity to paraffin sections of rabbit tissues. The results showed that an mAb reacted preferentially with late hypertrophic and calcified costal GC zones. The mAb also reacted with hypertrophic GC adjacent to bone that existed in sternum and femur, but not to other cartilages, including resting cartilage, articular cartilage, auricular cartilage, nasal cartilage, tracheal cartilage and meniscus cartilage, or with other tissues, including tendon, skin, muscles, lung, liver, heart, thymus, spleen, eye and gut. It reacted with a wider area of the GC zone when the sections were decalcified, although its reactivity with the extended area was much less intensive than that with late hypertrophic and calcified GC zones. On treatment of the sections with bacterial collagenase, neither the reactive area nor its intensity were changed, while when treated with trypsin the reactivity was lost. These results suggest the existence of a certain molecule which distinguishes GC (osteogenic cartilage) from other (non-osteogenic) cartilage. This mAb is a useful probe for distinguishing osteogenic cartilage from non-osteogenic cartilage, and for studying differentiation steps of cartilage cells in endochondral bone formation. The mAb can also be used as a probe for clinical and stored specimens because it reacts with decalcified and paraffin-embedded human specimens.

The repair of full-thickness articular cartilage defects. Immune responses to reparative tissue formed by allogeneic growth plate chondrocyte implants.

Growth plate cartilage cultivated in vitro was attached with a fibrin clot to a full-thickness articular cartilage defect on knee joints in allogeneic New Zealand rabbits. The healing of the defects was assessed by gross examination, light microscopy, and immunologic analysis for 24 weeks. Immunologic assessment of cell-mediated immunity, cytotoxicity of a humoral antibody by a 51chromium release assay, and immunofluorescence studies were carried out. During the first two weeks following grafting, healing was excellent in 11 of the 17 defects. From three to 24 weeks, 11 of 42 defects examined had good results. Host lymphocytes had accumulated around the allograft at two to 12 weeks. Most of the implanted cartilage grown in vitro died and was replaced by fibrous tissue. The immunologic studies suggested that the implanted cartilage began to degenerate two to three weeks after implantation partially because of a humoral immune response but more importantly because of cell-mediated cytotoxicity.

[Production of monoclonal antibody to chicken epiphyseal cartilage matrix vesicle metalloprotease to determine its localization].

Previous reports demonstrated that metalloprotease was isolated from matrix vesicles (MVs) of chicken epiphyseal cartilage. In this study, we produced the monoclonal antibody against MV metalloprotease (MVPase) and its location was studied by immunohistochemical technique. Isolated MVs were injected into Balb/c mice and spleen cells were collected for fusion with myeloma cells. A monoclonal hybrid cell producing antibody against MVPase was isolated. The immunoglobulin isotype was IgG1. On western blots, this antibody reacted with a single band protein of 33KDa which corresponded with purified MVPase. In the light immunohistochemical study, immuno reactions were observed in the mesenchymal cells of bone marrow at the lowest area in hypertrophic zone of the epiphyseal cartilage. Ultrastructural immunolocalization of protein A-gold particles revealing MVPase showed that MVs contained this enzyme, and in the calcifying area, gold particles were observed at the apatite needles of ruptured MVs. According to these observations, it is suggested that MVPase was already synthesized in undifferentiated marrow bone cells and this enzyme promotes the initial MV calcification and relates closely to the growth crystalline needles of apatite.

Expression of the cell proliferation-associated nuclear antigen reactive with the Ki-67 monoclonal antibody by cells of the skeletal system in humans and other species.

The cell proliferation-associated nuclear antigen recognized by the Ki-67 monoclonal antibody was detected immunohistochemically in cells from skeletal tissues of humans, sheep and rabbits. In each case the distribution of Ki-67-positive cells was identical and consistent with the known distribution of dividing cells in growing long bones. The antigen recognized by Ki-67 is detectable only after fixation in cold acetone. In the 5-day-old rabbit, labelling with tritiated thymidine [( 3H]TdR) in vivo, followed subsequently by a combination of Ki-67 immunohistochemistry and autoradiography for [3H]TdR, demonstrated that, in addition to the expression of Ki-67 by the majority of [3H]TdR labelled cells, other cells associated with proliferating populations also expressed the Ki-67 antigen. This suggests that the Ki-67 antibody identifies cycling cells in the skeleton in S-phase, as well as in other phases of the cell cycle. In Ki-67 reactive species, this method is a potentially useful tool for the study of cell proliferation in skeletal tissues in vivo and in vitro, having the advantage of not requiring prior labelling with DNA precursors.

Production and characterization of monoclonal antibodies against rabbit growth cartilage.

Growth cartilage (GC) cells of young rabbits were cultured in vitro and their homogenates were injected into mice. Hybridomas were prepared by the cell fusion technique between the myeloma cells and the spleen cells of the immunized mice. Monoclonal antibodies (MoAbs) were produced by the hybridomas in the peritoneal cavities of the mice, and some of these, temporarily named MoAbs A, B, D, N, P, and S, were studied. The localization of the antigens of each of the MoAbs in the GC or adjacent resting cartilage (RC) was examined by indirect fluorescent antibody staining. The molecular weight of the antigens was examined by immunoblot staining after SDS-polyacrylamide gel electrophoresis. MoAb A and MoAb N stained RC cells and GC cells, except calcified GC. MoAb B stained the hypertrophic and calcified GC, and matrices in the RC and proliferating GC. MoAb D stained the calcified GC. MoAb P and MoAb S stained the RC cells and the matrices in the GC, intensively in the hypertrophic GC and perichondrium. The molecular weights of the antigens of MoAbs A, P, and S were 40-70 KD, 35-40 KD and 30 KD, respectively.

Reactivity of normal rat epiphyseal chondrocytes with monoclonal antibodies recognizing different leucocyte markers.

The aim of the study was to test whether normal rat epiphyseal chondrocytes react with monoclonal antibodies (MoAbs) detecting different markers of lymphoid cells. For this purpose, isolated chondrocyte and, for comparison, splenocyte cytosmears were exposed to a battery of different MoAbs followed by indirect immunoperoxidase staining. As we were able to show, all chondrocytes reacted with OX17 MoAb detecting Class II (Ia) antigen encoded by the RT1.D subregion of rat major histocompatibility complex (MHC). However, unlike splenocytes they did not react with OX6 MoAb detecting the RT1.B encoded Class II molecule. Moreover, all chondrocytes were stained with W3/25 MoAb specific for the rat equivalent of human CD4 (T4) antigen and with W3/13 MoAb specific for rat leucocyte sialoglycoprotein. A positive reaction was also obtained with the antibody against the S-100 protein. By contrast, chondrocytes did not react with antibodies specific for all T (OX19) or B (HIS14) cells, rat CD8 (T8) equivalent, monocytes/macrophages (ED1, ED2), factor VIII (M616), or glial fibrillary acidic protein (Z334).

Cellular heterogeneity and insulin-like growth factor I immunoreactivity among epiphysial growth plate chondrocytes in the pig.

The localization of insulin-like growth factor I (IGF-I, also called somatomedin C) production in porcine epiphysial growth plates of the distal humerus was studied by immunohistochemistry. Counterstaining with Alcian blue-van Gieson demonstrated two cell types (blue and red cells) in the germinal (reserve), proliferating and hypertrophic zones; only those chondrocytes of the proliferative and hypertrophic zones that stained red were also immunoreactive to the antibody to IGF-I. The results indicate that there exists a functional heterogeneity among the chondrocytes of both the proliferative and hypertrophic zones of growth cartilage and that IGF-I is locally produced in only the red cells of these zones. Because the red cells of the germinal zone were not immunoreactive, the results suggest that the red cells of the germinal zone and the red cells of the proliferative and hypertrophic zones are also functionally distinct.