Expression of neurotrophins and their receptors in human bone marrow

Abstract

The expression of neurotrophins and their receptors, the low-affinity nerve growth factor receptor (p75LNGFR) and the Trk receptors (TrkA, TrkB, and TrkC), was investigated in human bone marrow from 16 weeks fetal age to adulthood. Using reverse transcription-polymerase chain reaction, all transcripts encoding for catalytic and truncated human TrkB or TrkC receptors were detected together with trkAI transcripts, whereas trkAII transcripts were found only in control nerve tissues. Transcripts for the homologue of the rat truncated TrkC(ic113) receptor were identified for the first time in human tissue. Stromal adventitial reticular cells were found immunoreactive for all neutrophin receptors. In contrast, hematopoietic cell types were not immunoreactive for p75LNGFR but showed immunoreactivity for one or several Trk receptors. TrkA immunoreactivity was found in immature erythroblasts. Catalytic TrkB immunoreactivity was observed in eosinophilic metamyelocytes and polymorphonuclear cells. Truncated TrkB immunoreactivity was found in erythroblasts and megacaryocytes. Immunoreactivity for both catalytic and truncated TrkC receptor was observed in promyelocytes, myelocytes, some polymorphonuclear cells and megacaryocytes. Neutrophin transcript levels appeared higher at fetal than at adult stages, no variation in Trk family transcript levels was observed. The local expression of neurotrophin genes suggests a wide range of paracrine and/or autocrine mode of action through their corresponding receptors within the bone marrow.

Figure 1.

Schematic diagram of the human trk receptor isoforms from Shelton et al. The primer sets location and the size of the expected RT-PCR products are summarized. ECD, extracellular domain; Kin, kinase domain; Trunc, truncated isoform.

Figure 2.

Immunohistochemical detection of p75^LNGFR and Trk in normal adult bone marrow: In the erythroblastic islets, a similar cytoplasmic and/or membranous TrkA IR was seen after immunostaining with both intracytoplasmic kinase TrkA (763) (A and C) and extracellular anti r-TrkA (B and D) antibodies. A truncated TrkB (C-13) IR was noted in erythroblasts and megaryocytes (E). In megacaryocytes, a strong TrkC IR was observed as a diffuse or focal cytoplasmic granular staining (arrowhead) (F). An intense cytoplasmic granular TrkC IR was also observed on immature promyelocytes and myelocytes (arrows) and in a few mature myeloid cells (F). A TrkA IR was observed in endothelial cells, in plasmocytes (arrowhead) and in adventitial reticular cells (ARC) (G). p75^LNGFR antibody also stained fibers in the adventitial coat of the larger capillaries (arrowhead) and ARC (arrows). Original magnifications, ×400 (A, B, E) and ×1000 (C, D, F-H).

Figure 3.

Immunocytochemical detection of p75^LNGFR and Trk in normal adult smears (APAAP method). Immunocytochemistry showed concordant results with IHC. Megacaryocytes (column 1) showed a diffuse intracytoplasmic truncated TrkB and an intense intracytoplasmic granular TrkC IR. In the granulocytic lineage (column 2), neutrophilic myelocytes, metamyelocytes and polymorphonuclear cells were TrkC immunoreactive. Erythroblasts (column 3) showed an intense TrkA IR (arrows) and, to a lesser degree, a truncated TrkB IR. p75^LNGFR IR was restricted to adventitial reticular cells (arrowheads). Original magnification, ×1000

Figure 4.

Trk transcripts expression in adult bone marrow. Amplification of regions of TrkA, TrkB and TrkC containing potential insert coding for extracellular or tyrosine kinase domain. The size of the RT-PCR product is indicated on the right. Each lane corresponds to the same bone marrow sample analyzed by RT-PCR. CP, caudate putamen RNA. MW: 100-bp ladder (Gibco BRL).

Figure 5.

Trk transcript expression in fetal bone marrow. Amplification of regions of TrkA, TrkB, and TrkC containing potential insert of extracellular domain or tyrosine kinase domain. The size of the RT-PCR product is indicated on the right. Each lane corresponds to the same bone marrow sample analyzed by RT-PCR. CP; caudate putamen RNA. MW: 100-bp ladder.

Figure 6.

Nucleotide sequence and deduced amino acid sequence of the human trkC extracellular RT-PCR-generated fragments. The boxed nucleotide sequence (24 bp) is missing on the shorter isoform (TrkCI). Nucleotides corresponding to the primers TrkC ECD 5′ and 3′ are underlined.

Figure 7.

A: Schematic diagram of the rat trkC truncated splice isoforms as reported by Valenzuela et al. Four alternative spliced exons (exons A, B, C, and D) encode for four different isoforms. TrkC ic158 is the only truncated isoform previously reported in human tissue. B: Human trkC truncated isoforms. Upper lane: nucleotide sequence of the two RT-PCR products (279 and 144 bp) generated with primers TrkC trunc by amplification of human bone marrow RNA. The boxed nucleotide sequence (135 bp) is missing on the shorter PCR product and is homologous to the rat exon B. Nucleotides corresponding to the primers TrkC trunc 5′ and 3′ are underlined. Predicted amino acid sequence is indicated for the two human TrkC ic158 and TrkC ic113 isoforms.

Figure 8.

Neurotrophin transcript distribution in human bone marrow. Amplification of NGF, BDNF, NT-3, and NT-4 transcripts in four adult and three fetal bone marrow samples which have been studied for Trk receptor expression (see Figures 4 and 5 ▶ ▶ ). Each lane corresponds to the same sample analyzed for the presence of the different transcripts. The size of the RT-PCR product is indicated on the right. Neurotrophin RT-PCR fragments are located on one exon. To ensure complete removal of potential DNA contamination, the amplification reaction was performed without the reverse transcription step (BDNF PCR without RT panel) for all samples except the caudate putamen RNA. The constitutively expressed Abelson (Abl) transcript was amplified by RT-PCR to ensure homogeneity within our sample. Adult bone marrow Abl amplification product was slightly less abundant than fetal and caudate putamen products. CP, caudate putamen RNA. MW: 100-bp ladder.