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We have previously reported that the expression of HC gp-39, a 39-kDa secretory glycoprotein and member of the chitinase protein family, is associated with late stages of monocyte to macrophage maturation. To allow further investigations of its unique expression pattern and to facilitate studies on the regulation of this "marker gene," we characterized the genomic organization of the HC gp-39 gene (HGMW-approved symbol CHI3L1) and cloned its 5'-proximal promotor region. The gene is composed of 10 exons distributed over an 8-kb region. Two transcriptional initiation sites located 82 and 126 nucleotides upstream of the ATG start codon were identified using primer extension and S1 nuclease protection assays. We sequenced 1.3 kb of the 5'-flanking region and identified a variety of putative regulatory elements.

The differentiation and activation of monocytes (MO) and monocytic cells is modulated by 1alpha,25-dihydroxyvitamin D3 (Vitamin D3). In order to investigate early effects on the differentiation process of MO, we used the mRNA Differential Display technology to identify genes that are induced in freshly isolated human blood MO cultured for 4 hours with Vitamin D3. A cDNA fragment was isolated and Northern analysis confirmed a low expression of this cDNA at about 1,4 kb in MO which was increased by the addition of Vitamin D3. Using the rapid amplification of cDNA Ends (RACE)-PCR we got a transcript (DDVit 1) of a length of 1251 bp containing an open reading frame that encodes a putative 16,5 kD protein. Database search revealed an identity with a possible enterocyte differentiation promoting factor with a length of 1177 bp that has not been further characterized. Therefore DDVit 1 may be a differentiation promoting factor for the monocytic lineage. Further investigations will clarify the role of this protein in the differentiation process of MO.

Maturation of cells of the mononuclear phagocyte lineage from bone marrow precursors to tissue macrophages (MAC) via circulating blood monocytes (MO) is a multistep process only partially understood. Similarly, MAC differentiation can be observed if MO are cultured in vitro. In an attempt to further characterize molecular changes occurring during this process we carried out differential screening of a MO-derived MAC cDNA library using MO and MAC cDNA. After subcloning and confirmation by a second round of screening, partial sequencing of 41 cDNA clones was performed. In 33 clones the sequences of 7 different previously identified cDNAs were found. The mRNA expression of two of the corresponding genes (apolipoprotein E, ferritin light chain) is already known to be up-regulated during MAC maturation. For one gene (cathepsin B), a specific up-regulation of mRNA expression could be shown corresponding to previous protein data. For four genes [human cartilage glycoprotein (HC-gp39), osteopontin, type IV collagenase, and tryptophanyl-tRNA synthetase] the specific expression in MAC versus MO was previously unknown but could be confirmed by the use of Northern blot analysis. Of these genes, HC-gp39 is especially interesting because it is only expressed during the late stages of MAC differentiation.

Here we report on an experimental system for generating TAM in vitro by culturing human MO and MO-derived macrophages (MAC) within 3-dimensional multicellular tumor spheroids (MCS). MO as well as MO-derived MAC migrate into tumor spheroids and spread throughout the entire spheroid within 16 hr. In contrast, fibroblast-spheroids were not infiltrated. The regular expression of MAC maturation-associated antigens on infiltrating MO was suppressed within MCS of the undifferentiated bladder carcinoma line J82 with regard to carboxypeptidase M (CPM), MAX.3 antigen and CD105. However, MAC within spheroids of highly differentiated papillary RT4 cells failed only the single antigen CD51, whereas MAC expressed the complete maturation-associated phenotype within non-tumorigenic HCV29 spheroids. Interestingly, the suppressive effect of J82 carcinoma cells could only be observed in 3-dimensional but not in monolayer cultures. The J82-MCS induced suppression of CPM and MAX.3 expression was only seen to be operative on infiltrating blood MO: MO first differentiated for 2 days and subsequently co-cultured with J82-MCS showed normal expression of MAX.3 and CPM within the spheroid. Besides the modulation of MAC phenotype, the cytokine response of intraspheroidal MAC was analyzed: upon co-culture MO secreted high IL-1beta and IL-6 but low amounts of TNF-alpha as compared to MAC. This MO typical cytokine pattern remained constant for up to 8 days in culture, again indicating a disturbed MO to MAC maturation within tumor spheroids. In conclusion, a 3-dimensional interaction with tumor cells in vitro results in significant changes in the phenotype and function of the spheroid-associated MO and MAC.

It is well known that adherence of monocytes (MO) to extracellular matrix substrates or tissue culture plastic activates these cells and induces the expression of a multitude of genes. Especially, it was described, that MO are primed by cell adhesion to produce higher amounts of some cytokines, e.g. interleukin (IL)-8 and tumor necrosis factor alpha (TNF-alpha). In order to investigate adherence-induced effects upon cytokine production, we seeded MO into tissue cultures and stimulated cells by lipopolysaccharide (LPS) simultaneously or at later time points. An increasing time-lag between cell adhesion and LPS-stimulation led to differential effects upon cytokine production: whereas TNF was upregulated (in accordance with reports by others), granulocyte colony-stimulating factor (G-CSF) was considerably down-regulated. In contrast, G-CSF production did not change, when cells were kept under non-adherent conditions in whole blood. In adherent cultures down-regulation of G-CSF could already be observed after two hours with a maximum after 24 h and was paralleled by a much lower abundance of G-CSF mRNA. Adhesion induced a significant suppression of G-CSF comparable to MO, if mature macrophages derived from MO in vitro were examined. Furthermore, two other cytokines, granulocyte-macrophage (GM)-CSF and IL-6, were also down-regulated following adhesion. In conclusion, activation of mononuclear phagocytes by adhesion can lead to "priming" for the production of some cytokines and at the same time to "silencing" for the production of others.

Differentiation of tissue macrophages from bone marrow progenitor cells via circulating blood monocytes is a complex and multistep process. In order to analyze macrophage maturation on the molecular level we used the method of mRNA differential display to identify novel genes that are preferentially expressed in mature, in vitro differentiated macrophages. A PCR-fragment could be isolated that was amplified from macrophages, but not from freshly isolated monocytes, and macrophage-specific expression was confirmed by Northern blot analysis. Several corresponding cDNA clones could be isolated from a macrophage cDNA library, covering a nucleotide sequence of 2.5 kb. Nucleotide sequence of this cDNA showed no homology to known genes. The sequence reveals an open reading frame that codes for a 238 amino acid hydrophobic protein with seven potential transmembrane domains, suggesting a possible role as a receptor or an ion channel.

It has been well established that human mononuclear phagocytes have the capacity to produce 1,25-dihydroxy-vitamin D3 [1,25(OH)3D3] and express the vitamin D receptor (VDR). However, 1 alpha-hydroxylase activity and VDR receptor expression during differentiation of monocytes (MO) into mature macrophages (MAC) have not been previously examined. The in vitro maturation of blood MO can serve as a model for the in vivo transformation of immature blood MO into MAC. Here, when cultured in the presence of serum, MO undergo characteristic changes in morphology, antigenic phenotype, and functional activity consistent with their differentiation into MAC. We serially measured 1,25(OH)2D3 and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] synthesis, specific [3H]-1,25(OH)2D3 binding, and VDR mRNA levels during in vitro maturation of MO into MAC and correlated these functions with maturation-associated changes in the phenotype (MAX.1 and CD71) and secretory repertoire (interleukin-1 beta [IL-1 beta], neopterin) of the cells. MO showed only little conversion of 25-(OH)D3 into 1,25(OH)2D3 (1.4 +/- 0.4 pmol/10(6) cells/6 h, n = 5) that increased gradually during maturation into MAC at day 8 of culture (5.3 +/- 4.3 pmol/10(6) cells/6 h, n = 5). Interferon-gamma (IFN-gamma) increased baseline 1,25(OH)2D3-synthesis approximately twofold during all phases of differentiation. The time course of increased 1,25(OH)2D3-synthesis correlated with enhanced secretion of neopterin and expression of MAX.1 and CD71. The addition of exogenous 1,25(OH)2D3 did not influence constitutive 1,25(OH)2D3 synthesis, but IFN-gamma-stimulated production was suppressed to baseline levels. Exogenous 1,25(OH)2D3 also stimulated 24,25(OH)2D3 synthesis in freshly isolated MO (from 1.0 +/- 0.8 pmol/6 h to 5.6 +/- 0.9 pmol), whereas matured MAC showed no 24,25(OH)2D3 synthesis. Furthermore, we examined the expression of the VDR during the differentiation process. VDR mRNA and protein were constitutively expressed in MO, whereas VDR was downregulated in mature MAC on both the mRNA and protein levels. Homologous upregulation of VDR protein by 1,25(OH)2D3 occurred in MO and, to a lesser degree, in MAC. In contrast, VDR mRNA concentrations were not influenced by 1,25(OH)2D3. Taken together, our results show that MO into MAC differentiation in vitro is associated with (1) an enhanced capacity to synthesize 1,25(OH)2D3, (2) a loss of 24,25(OH)2D3-synthesizing activity, and (3) a decrease in the expression of VDR mRNA and protein. Because 1,25(OH)2D3 was shown to induce differentiation of MO into MAC, our data sugest an autoregulatory mechanism of MO/MAC generation by 1,25(OH)2D3.

Macrophages (MAC) are important effector cells of the immune system. They arise from circulating blood monocytes (MO), which undergo further maturation upon leaving the vasculature and migrating into the various tissues and body cavities. A similar differentiation process can be followed in vitro when monocytes are cultured in the presence of serum. In this study, different factors and serum proteins, either alone or in combination, were tested for their ability to promote the survival and/or maturation of blood MO in the absence of serum. Elutriation-purified MO cultured for 8 days on hydrophobic teflon foils in the presence of 5% human serum differentiated into large, well-spread MAC, whereas in the absence of serum, MO rapidly died. The serum-induced maturation of MAC was accompanied by a strong expression of CD16, CD14 and MAX antigens. Secretion of TNF-alpha and neopterin increased about 10-fold as compared with freshly isolated MO. The replacement of serum by either M-CSF (100 ng/ml) or immunoglobulin (0.5-5 mg/ml) had a marked effect on MO survival (about 50% of serum-cultured MO), but cells were smaller, less spread out and had low expression of CD16, CD14 and MAX antigens. Their functional competence in terms of TNF-alpha and neopterin release was reduced to 10-20% as compared with MAC cultured in the presence of serum.(ABSTRACT TRUNCATED AT 250 WORDS)