Macrophage function in the elderly and impact on injury repair and cancer.

Older age is associated with deteriorating health, including escalating risk of diseases such as cancer, and a diminished ability to repair following injury. This rise in age-related diseases/co-morbidities is associated with changes to immune function, including in myeloid cells, and is related to immunosenescence. Immunosenescence reflects age-related changes associated with immune dysfunction and is accompanied by low-grade chronic inflammation or inflammageing. This is characterised by increased levels of circulating pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1ß and IL-6. However, in healthy ageing, there is a concomitant age-related escalation in anti-inflammatory cytokines such as transforming growth factor-ß1 (TGF-ß1) and IL-10, which may overcompensate to regulate the pro-inflammatory state. Key inflammatory cells, macrophages, play a role in cancer development and injury repair in young hosts, and we propose that their role in ageing in these scenarios may be more profound. Imbalanced pro- and anti-inflammatory factors during ageing may also have a significant influence on macrophage function and further impact the severity of age-related diseases in which macrophages are known to play a key role. In this brief review we summarise studies describing changes to inflammatory function of macrophages (from various tissues and across sexes) during healthy ageing. We also describe age-related diseases/co-morbidities where macrophages are known to play a key role, focussed on injury repair processes and cancer, plus comment briefly on strategies to correct for these age-related changes.

F2-isoprostanes affect macrophage migration and CSF-1 signalling.

F2-isoprostanes (F2-IsoP) are formed in vivo via free radical peroxidation of arachidonic acid. Enhanced oxidative stress is implicated in the development of atherosclerosis in humans and F2-IsoP have been detected in atherosclerotic plaque. Colony stimulating factor-1 (CSF-1) is essential to macrophage survival, proliferation and differentiation and has been detected in human atherosclerotic plaques. Accumulation of macrophages within the vascular wall is an important component of atherosclerosis but little is known about the effect of F2-IsoP on the migration of these cells. Our aim was to examine the effect of free and lipid-bound 15-F2t-isoprostane (15-F2t-IsoP) on macrophage migration and investigate the signalling pathways involved. Mouse macrophages (cell line BAC1.2F5) were pre-incubated with 15-F2t-IsoP (free, bound to cholesterol or monoacylglycerol or within oxidized phospholipid) and cell migration was assessed using chemotaxis towards CSF-1 in Boyden chambers. Migration was also measured using the wound healing assay with primary mouse bone marrow derived macrophages. We showed that 15-F2t-IsoP dose-dependently inhibited BAC1.2F5 macrophage spreading and adhesion but stimulated their migration towards CSF-1, with maximum effect at 10 µM. Analysis of CSF-1 stimulated signalling pathways in BAC1.2F5 macrophages showed that phosphorylation of Akt, a key mediator of cell migration, and one of its regulators, the mTORC2 component, Rictor, was significantly decreased. In contrast, phosphorylation of the adhesion kinases, FAK and Pyk2, and the adhesion scaffold protein, paxillin, was enhanced after treatment with 15-F2t-IsoP. Mouse bone marrow macrophages were transfected with FAK or Pyk2 small interfering RNA (siRNA) to examine the role of FAK and Pyk2 in 15-F2t-IsoP signalling. Pyk2 silencing inhibited 15-F2t-IsoP-induced reduction in cell area and phospho-paxillin adhesion numbers. The size distribution of adhesions in the presence of 15-F2t-IsoP was also affected by Pyk2 silencing and there was a trend for Pyk2 silencing to reduce 15-F2t-IsoP-stimulated macrophage migration. These results demonstrate that 15-F2t-IsoP affects macrophage adhesions and migration, which are integral components of macrophage involvement in atherosclerosis.

Protein tyrosine phosphatase phi regulates paxillin tyrosine phosphorylation and mediates colony-stimulating factor 1-induced morphological changes in macrophages.

Removal of colony-stimulating factor 1 (CSF-1) causes macrophages to round up and to increase their expression of protein tyrosine phosphatase phi (PTP phi). This is accompanied by the disruption of focal complexes and the formation of ruffles. Here we have overexpressed wild-type (WT) PTP phi and a phosphatase-inactive (C325S) mutant in a macrophage cell line in the presence and absence of CSF-1. In the presence of CSF-1, WT PTP phi induces cell rounding and ruffle formation, while C325S PTP phi has no effect. In contrast, in CSF-1-starved cells, C325S PTP phi behaves in a dominant negative fashion, preventing rounding and ruffling. Furthermore, C325S PTP phi increases adhesion in cycling cells, while WT PTP phi enhances motility. In WT PTP phi-overexpressing cells, the focal contact protein paxillin is selectively depleted from focal complexes and specifically dephosphorylated on tyrosine. In contrast, paxillin is hyperphosphorylated in C325S PTP phi-expressing cells. Moreover, a complex containing PTP phi, paxillin, and a paxillin-associated tyrosine kinase, Pyk2, can be immunoprecipitated from macrophage lysates, and the catalytic domain of PTP phi selectively binds paxillin and Pyk2 in vitro. Although PTP phi and Pyk2 do not colocalize with paxillin in focal complexes, all three proteins are colocalized in dorsal ruffles. The results suggest that paxillin is dephosphorylated by PTP phi in dorsal ruffles, using Pyk2 as a bridging molecule, resulting in a reduced pool of tyrosine-phosphorylated paxillin available for incorporation into focal complexes, thereby mediating CSF-1 regulation of macrophage morphology, adhesion, and motility.

Regulation of mouse podocyte process dynamics by protein tyrosine phosphatases rapid communication.

BACKGROUND: Effacement of podocyte foot processes occurs early in many glomerular diseases associated with proteinuria and is accompanied by a reorganization of the actin cytoskeleton. The molecular mechanisms regulating these structural changes are poorly understood. METHODS: To address these questions, we analyzed the effect of the polycation, protamine sulfate (PS), and puromycin aminonucleoside (PA) on the morphology, cytoskeleton, and tyrosine phosphorylation of differentiated process-bearing cultured podocytes. RESULTS: PS and PA induced similar profound morphological alterations, including retraction and detachment of podocyte processes from the extracellular matrix (ECM). The effects of PS occurred within six hours, whereas PA showed its most severe effects after 72 hours. Structural changes included reorganization of the actin cytoskeleton and focal contacts and were accompanied by an increase in tyrosine phosphorylation. The same effects were induced by application of vanadate, an inhibitor of protein tyrosine phosphatases (PTPs), suggesting that PTPs regulate podocyte process structure. Since disruption of the actin cytoskeleton with cytochalasin B protected the cells from PS-induced effacement and detachment, cytoplasmic PTPs were implicated in these events. Using reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated the expression of four cytoplasmic PTPs in podocytes: SHP-2, PTP-PEST, PTP-1B, and PTP-36. CONCLUSIONS: These studies indicate an important role for cytoplasmic PTPs as regulators of podocyte process dynamics. Future studies will aim at restoring the normal foot process architecture of podocytes in glomerular diseases associated with proteinuria by modulating the activity of cytoplasmic PTPs.

Biology and action of colony--stimulating factor-1.

Colony-stimulating factor-1 (CSF-1), also known as macrophage colony-stimulating factor, controls the survival, proliferation, and differentiation of mononuclear phagocytes and regulates cells of the females reproductive tract. It appears to play an autocrine and/or paracrine role in cancers of the ovary, endometrium, breast, and myeloid and lymphoid tissues. Through alternative mRNA splicing and differential post-translational proteolytic processing, CSF-1 can either be secreted into the circulation as a glycoprotein or chondroitin sulfate-containing proteoglycan or be expressed as a membrane-spanning glycoprotein on the surface of CSF-1-producing cells. Studies with the op/op mouse, which possesses an inactivating mutation in the CSF-1 gene, have established the central role of CSF-1 in directly regulating osteoclastogenesis and macrophage production. CSF-1 appears to preferentially regulate the development of macrophages found in tissues undergoing active morphogenesis and/or tissue remodeling. These CSF-1 dependent macrophages may, via putative trophic and/or scavenger functions, regulate characteristics such as dermal thickness, male fertility, and neural processing. Apart from its expression on mononuclear phagocytes and their precursors, CSF-1 receptor (CSF-1R) expression on certain nonmononuclear phagocytic cells in the female reproductive tract and studies in the op/op mouse indicate that CSF-1 plays important roles in female reproduction. Restoration of circulating CSF-1 to op/op mice has preliminarily defined target cell populations that are regulated either humorally or locally by the synthesis of cell-surface CSF-1 or by sequestration of the CSF-1 proteoglycan. The CSF-1R is a tyrosine kinase encoded by the c-fms proto-oncogene product. Studies by several groups have used cells expressing either the murine or human CSF-1R in fibroblasts to pinpoint the requirement of kinase activity and the importance of various receptor tyrosine phosphorylation sites for signaling pathways stimulated by CSF-1. To investigate post-CSF-1R signaling in the macrophage, proteins that are rapidly phosphorylated on tyrosine in response to CSF-1 have been identified, together with proteins associated with them. Studies on several of these proteins, including protein tyrosine phosphates 1C, the c-cbl proto-oncogene product, and protein tyrosine phosphatase-phi are discussed.

A heteromorphic protein-tyrosine phosphatase, PTP phi, is regulated by CSF-1 in macrophages.

A novel protein-tyrosine phosphatase, PTP phi, was cloned from a murine macrophage cDNA library. As a result of alternative splicing, macrophage PTP phi mRNAs are predicted to encode two membrane-spanning molecules and a cytosolic enzyme with identical catalytic domains. The membrane-spanning forms differ in the juxtamembrane region, while a start codon downstream of this region is utilized in the translation of the putative cytosolic form. Expression of PTP phi mRNA is low and restricted to macrophage cell lines, macrophage-rich tissues, and brain, kidney, and heart. The mRNA in macrophages and heart is approximately 2.8 kilobases (kb). However, a approximately 5.5-kb transcript in brain and kidney indicates a fourth isoform encoding a large extracellular domain. The approximately 5.5-kb PTP phi brain mRNA encodes the mouse homolog of GLEPP1, a recently reported glomerular epithelial protein. The level of expression of the mRNA encoding the cytosolic form was very low, and only the membrane-spanning proteins (43 and 47 kDa) could be detected in macrophages. Following addition of colony stimulating factor-1 to quiescent BAC1.2F5 macrophages, PTP phi mRNA and protein were down-regulated. The restricted expression of the shorter isoforms of PTP phi and their regulation by colony stimulating factor-1 in macrophages suggest that PTP phi may play a role in mononuclear phagocyte survival, proliferation, and/or differentiation.

Protein tyrosine phosphatase-1C is rapidly phosphorylated in tyrosine in macrophages in response to colony stimulating factor-1.

An approximately 64-kDa cytoplasmic protein is rapidly phosphorylated in tyrosine in the response of macrophages to colony stimulating factor-1. To identify this protein, BAC1.2F5 macrophages were incubated with or without colony stimulating factor-1, the phosphotyrosine-containing portion of their cytosolic fractions subjected to size exclusion chromatography, and the 45-70-kDa fraction further fractionated by reverse phase high pressure liquid chromatography (RP-HPLC). Tryptic peptides of pooled RP-HPLC fractions from stimulated cells (containing the approximately 64-kDa protein and an approximately 54-kDa protein) and from unstimulated cells (containing the approximately 54-kDa protein alone), were sequenced directly. All seven readable sequences of 8 sequenceable peptides present uniquely in the stimulated fraction were present in the sequence of the src homology 2 domain-containing protein tyrosine phosphatase-1C (PTP-1C). The identity of the approximately 64-kDa protein was confirmed by Western blotting with an antibody raised to a PTP-1C peptide. The rapid, growth factor-induced tyrosine phosphorylation of PTP-1C suggests that it may be involved in very early events in growth factor signal transduction.

Mitochondrial gene sequences show fungal homology for Pneumocystis carinii.

A 6.8 kilobase fragment of mitochondrial DNA from Pneumocystis carinii encodes for apocytochrome b, NADH dehydrogenase subunits 1, 2, 3, and 6, cytochrome oxidase subunit II, and the small subunit of ribosomal RNA. Comparative sequence analysis with a series of organisms representative of the fungal and protozoan groups shows that P. carinii has, consistently, an average similarity of 60% with the fungi but only 20% with the protozoa. The data indicate homology with the fungi for this opportunistic pathogen.

Amplification of mitochondrial ribosomal RNA sequences from Pneumocystis carinii DNA of rat and human origin.

Pneumocystis carinii specific DNA sequences have been cloned from the experimental rat model. The sequence of the gene coding for the large subunit of mitochondrial ribosomal RNA has been used to construct P. carinii specific oligonucleotide primers for the polymerase chain reaction. These oligonucleotides produced amplification of specific sequences from both P. carinii infected rat and human lung samplings, but none from a range of other organisms including potential pulmonary pathogens. Comparison of the sequence of amplified products from the infected rats and humans demonstrated limited but consistent differences between P. carinii from these two hosts and allowed for the construction of a human specific internal oligonucleotide. The application of the specific oligonucleotides for DNA amplification and subsequent Southern hybridisation affords extremely sensitive and specific detection of P. carinii in human samples, which may be applicable to both epidemiological research and clinical studies.

Detection of Pneumocystis carinii with DNA amplification.

Oligonucleotide primers and probes were used in the polymerase chain reaction to amplify Pneumocystis carinii specific DNA sequences from alveolar lavage samples from 47 diagnostic bronchoscopies. No P carinii DNA was found in lavage from 10 immunocompetent patients; only low levels were found in 3 of 13 samples from immunosuppressed individuals without P carinii pneumonia (PCP), and the highest levels, readily demonstrated by simple ethidium bromide staining, were found in all of 16 samples from immunosuppressed patients with PCP confirmed by means of standard silver staining and in 4 from patients with clinical PCP but negative silver staining. DNA amplification provides a highly sensitive and specific technique for the identification of P carinii that should be valuable in epidemiological studies on this parasitic infection and in diagnosis.

Importance of beta 2-adrenoceptor stimulation in the suppression of intradermal antigen challenge by adrenaline.

1. Seven atopic subjects received two injections of antigen and one of saline intradermally in the back on each of 4 separate days. They were pretreated with four different drug combinations: (a) adrenaline 0.3 mg subcutaneously over the deltoid muscle (b) subcutaneous adrenaline preceded by 5 mg of the specific beta 2-adrenoceptor antagonist ICI 118,551 orally (c) 8 mg of salbutamol orally (d) placebo. Tablets were given 2 h before and subcutaneous injections 15 min before the intradermal injections of saline and antigen. 2. The median flare response to intradermal low dose antigen and high dose antigen after pretreatment with adrenaline was 4% and 49% of the response seen following pretreatment with placebo (P less than 0.001). When adrenaline was preceded by ICI-118,551, the corresponding median flare responses were 2% and 44% (P less than 0.001) of the placebo response. The flare response after pretreatment with salbutamol was not significantly different from placebo. 3. Adrenaline suppressed the median weal response to the higher dose of antigen to 52% of the response after pretreatment with placebo (P less than 0.05). This suppression by adrenaline was blocked by pretreatment with ICI 118,551. The median weal response after the highest dose of antigen was suppressed by salbutamol to 66% of the response seen after placebo, although this was not significant even when a further three subjects were studied with either salbutamol or placebo.(ABSTRACT TRUNCATED AT 250 WORDS)

The importance of bradykinin and histamine in the skin response to antigen.

On three separate occasions 12 atopic subjects were injected intradermally with two doses of antigen and one of saline as control. Pretreatment with terfenadine 60 mg orally significantly inhibited the flare response to both the lower dose of antigen and to saline (P less than 0.05). Ingestion of enalapril 5 mg orally 3 h before increased the flare response to both doses of antigen. Neither enalapril nor terfenadine affected the weal response when compared with placebo. Both endogenous histamine and bradykinin appear to be released during the intradermal flare response but are not important in the weal reaction to antigen.