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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) [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Although immunotherapy has revolutionized cancer treatment, many immunogenic tumors remain refractory to treatment. This can be largely attributed to an immunologically "cold" tumor microenvironment characterized by an accumulation of immunosuppressive myeloid cells and exclusion of activated T cells. Here, we demonstrate that genetic ablation or therapeutic inhibition of the myeloid-specific hematopoietic cell kinase (HCK) enables activity of antagonistic anti-programmed cell death protein 1 (anti-PD1), anti-CTLA4, or agonistic anti-CD40 immunotherapies in otherwise refractory tumors and augments response in treatment-susceptible tumors. Mechanistically, HCK ablation reprograms tumor-associated macrophages and dendritic cells toward an inflammatory endotype and enhances CD8 + T cell recruitment and activation when combined with immunotherapy in mice. Meanwhile, therapeutic inhibition of HCK in humanized mice engrafted with patient-derived xenografts counteracts tumor immunosuppression, improves T cell recruitment, and impairs tumor growth. Collectively, our results suggest that therapeutic targeting of HCK activity enhances response to immunotherapy by simultaneously stimulating immune cell activation and inhibiting the immunosuppressive tumor microenvironment.

Increased cancer incidence occurs with the emergence of immunosenescence, highlighting the indispensability of the immune system in preventing cancer and its dysregulation with aging. Tumor-associated macrophages (TAMs) are often present in high numbers and are associated with poor clinical outcomes in solid cancers, including mesothelioma. Monocytes and macrophages from the bone marrow and spleen can respond to tumor-derived factors, such as CSF-1, and initiation of the CSF-1R signaling cascade results in their proliferation, differentiation, and migration to the tumor. Age-related changes occur in monocytes and macrophages in terms of numbers and function, which in turn can impact tumor initiation and progression. Whether this is due to changes in CSF-1R expression with aging is currently unknown and was investigated in this study. We examined monocytes and macrophages in the bone marrow and spleen during healthy aging in young (3-4 months) and elderly (20-24 months) female C57BL/6J mice. Additionally, changes to these tissues and in TAMs were examined during AE17 mesothelioma tumor growth. Healthy aging resulted in an expansion of Ly6C high monocytes and macrophages in the bone marrow and spleen. CSF-1R expression levels were reduced in elderly splenic macrophages only, suggesting differences in CSF-1R signaling between both cell type and tissue site. In tumor-bearing mice, Ly6C high monocytes increased with tumor growth in the spleen in the elderly and increased intracellular CSF-1R expression occurred in bone marrow Ly6C high monocytes in elderly mice bearing large tumors. Age-related changes to bone marrow and splenic Ly6C high monocytes were reflected in the tumor, where we observed increased Ly6C high TAMs earlier and expansion of Ly6C low TAMs later during AE17 tumor growth in the elderly compared to young mice. F4/80 high TAMs increased with tumor growth in both young and elderly mice and were the largest subset of TAMs in the tumor. Together, this suggests there may be a faster transition of Ly6C high towards F4/80 high TAMs with aging. Amongst TAM subsets, expression of CSF-1R was lowest in F4/80 high TAMs, however Ly6C low TAMs had higher intracellular CSF-1R expression. This suggests downstream CSF-1R signaling may vary between macrophage subsets, which can have implications towards CSF-1R blockade therapies targeting macrophages in cancer., Competing Interests: DN acts as a non-salaried Chief Scientific Officer for Selvax. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Duong, Pixley, Nelson and Jackaman.)

The mesothelium when first described was thought to function purely as a non-adhesive surface to facilitate intracoelomic movement of organs. However, the mesothelium is now recognized as a dynamic cellular membrane with many important functions that maintain serosal integrity and homeostasis. For example, mesothelial cells interact with and help regulate the body's inflammatory and immune system following infection, injury, or malignancy. With recent advances in our understanding of checkpoint molecules and the advent of novel immunotherapy approaches, there has been an increase in the number of studies examining mesothelial and immune cell interaction, in particular the role of these interactions in malignant mesothelioma. This review will highlight some of the recent advances in our understanding of how mesothelial cells help regulate serosal immunity and how in a malignant environment, the immune system is hijacked to stimulate tumor growth. Ways to treat mesothelioma using immunotherapy approaches will also be discussed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Persistent activation of the latent transcription factor STAT3 is observed in gastric tumor epithelial and immune cells and is associated with a poor patient prognosis. Although targeting STAT3-activating upstream kinases offers therapeutically viable targets with limited specificity, direct inhibition of STAT3 remains challenging. Here we provide functional evidence that myeloid-specific hematopoietic cell kinase (HCK) activity can drive STAT3-dependent epithelial tumor growth in mice and is associated with alternative macrophage activation alongside matrix remodeling and tumor cell invasion. Accordingly, genetic reduction of HCK expression in bone marrow-derived cells or systemic pharmacologic inhibition of HCK activity suppresses alternative macrophage polarization and epithelial STAT3 activation, and impairs tumor growth. These data validate HCK as a molecular target for the treatment of human solid tumors harboring excessive STAT3 activity., (©2020 American Association for Cancer Research.)

Migratory macrophages play critical roles in tissue development, homeostasis and disease, so it is important to understand how their migration machinery is regulated. Whole-transcriptome sequencing revealed that CSF-1-stimulated differentiation of bone marrow-derived precursors into mature macrophages is accompanied by widespread, profound changes in the expression of genes regulating adhesion, actin cytoskeletal remodeling and extracellular matrix degradation. Significantly altered expression of almost 40% of adhesion genes, 60-86% of Rho family GTPases, their regulators and effectors and over 70% of extracellular proteases occurred. The gene expression changes were mirrored by changes in macrophage adhesion associated with increases in motility and matrix-degrading capacity. IL-4 further increased motility and matrix-degrading capacity in mature macrophages, with additional changes in migration machinery gene expression. Finally, siRNA-induced reductions in the expression of the core adhesion proteins paxillin and leupaxin decreased macrophage spreading and the number of adhesions, with distinct effects on adhesion and their distribution, and on matrix degradation. Together, the datasets provide an important resource to increase our understanding of the regulation of migration in macrophages and to develop therapies targeting disease-enhancing macrophages., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Most cancers emerge in the elderly, including lung cancer and mesothelioma, yet the elderly remain an underrepresented population in pre-clinical cancer studies and clinical trials. The immune system plays a critical role in the effectiveness of many anti-cancer therapies in young hosts via tumor-specific T cells. However, immunosuppressive macrophages can constitute up to 50% of the tumor burden and impair anti-tumor T cell activity. Altered macrophage phenotype and function during aging may further impact anti-tumor T cell responses. Yet, the impact of macrophages on anti-tumor T cell responses and immunotherapy in the elderly is unknown. Therefore, we examined macrophages and their interaction with T cells in young (3 months) and elderly (20-24 months) AE17 mesothelioma-bearing female C57BL/6J mice during tumor growth. Mesothelioma tumors grew faster in elderly compared with young mice, and this corresponded with an increase in tumor-associated macrophages. During healthy aging, macrophages increase in bone marrow and spleens suggesting that these sites have an increased potential to supply cancer-promoting macrophages. Interestingly, in tumor-bearing mice, bone marrow macrophages increased proliferation whilst splenic macrophages had reduced proliferation in elderly compared with young mice, and macrophage depletion using the F4/80 antibody slowed tumor growth in young and elderly mice. We also examined responses to treatment with intra-tumoral IL-2/anti-CD40 antibody immunotherapy and found it was less effective in elderly (38% tumor regression) compared to young mice (90% regression). Tumor-bearing elderly mice decreased in vivo anti-tumor cytotoxic T cell activity in tumor draining lymph nodes and spleens. Depletion of macrophages using F4/80 antibody in elderly, but not young mice, improved IL-2/anti-CD40 immunotherapy up to 78% tumor regression. Macrophage depletion also increased in vivo anti-tumor T cell activity in elderly, but not young mice. All the tumor-bearing elderly (but not young) mice had decreased body weight (i.e., exhibited cachexia), which was greatly exacerbated by immunotherapy; whereas macrophage depletion prevented this immunotherapy-induced cachexia. These studies strongly indicate that age-related changes in macrophages play a key role in driving cancer cachexia in the elderly, particularly during immunotherapy, and sabotage elderly anti-tumor immune responses.

F 2 -isoprostanes (F 2 -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 F 2 -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 F 2 -IsoP on the migration of these cells. Our aim was to examine the effect of free and lipid-bound 15-F 2t -isoprostane (15-F 2t -IsoP) on macrophage migration and investigate the signalling pathways involved. Mouse macrophages (cell line BAC1.2F5) were pre-incubated with 15-F 2t -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-F 2t -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-F 2t -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-F 2t -IsoP signalling. Pyk2 silencing inhibited 15-F 2t -IsoP-induced reduction in cell area and phospho-paxillin adhesion numbers. The size distribution of adhesions in the presence of 15-F 2t -IsoP was also affected by Pyk2 silencing and there was a trend for Pyk2 silencing to reduce 15-F 2t -IsoP-stimulated macrophage migration. These results demonstrate that 15-F 2t -IsoP affects macrophage adhesions and migration, which are integral components of macrophage involvement in atherosclerosis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The ability of macrophages to respond to chemoattractants and inflammatory signals is important for their migration to sites of inflammation and immune activity and for host responses to infection. Macrophages differentiated from the bone marrow (BM) of UV-irradiated mice, even after activation with LPS, migrated inefficiently toward CSF-1 and CCL2. When BM cells were harvested from UV-irradiated mice and transplanted into naive mice, the recipient mice (UV-chimeric) had reduced accumulation of elicited monocytes/macrophages in the peritoneal cavity in response to inflammatory thioglycollate or alum. Macrophages differentiating from the BM of UV-chimeric mice also had an inherent reduced ability to migrate toward chemoattractants in vitro, even after LPS activation. Microarray analysis identified reduced reticulon-1 mRNA expressed in macrophages differentiated from the BM of UV-chimeric mice. By using an anti-reticulon-1 Ab, a role for reticulon-1 in macrophage migration toward both CSF-1 and CCL2 was confirmed. Reticulon-1 subcellular localization to the periphery after exposure to CSF-1 for 2.5 min was shown by immunofluorescence microscopy. The proposal that reduced reticulon-1 is responsible for the poor inherent ability of macrophages to respond to chemokine gradients was supported by Western blotting. In summary, skin exposure to erythemal UV radiation can modulate macrophage progenitors in the BM such that their differentiated progeny respond inefficiently to signals to accumulate at sites of inflammation and immunity., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Monocytes/macrophages differentiating from bone marrow (BM) cells pulsed for 2 hours at 37°C with a stabilized derivative of prostaglandin E 2 , 16,16-dimethyl PGE 2 (dmPGE 2 ), migrated less efficiently toward a chemoattractant than monocytes/macrophages differentiated from BM cells pulsed with vehicle. To confirm that the effect on BM cells was long lasting and to replicate human BM transplantation, chimeric mice were established with donor BM cells pulsed for 2 hours with dmPGE 2 before injection into marrow-ablated congenic recipient mice. After 12 weeks, when high levels (90%) of engraftment were obtained, regenerated BM-derived monocytes/macrophages differentiating in vitro or in vivo migrated inefficiently toward the chemokines colony-stimulating factor-1 (CSF-1) and chemokine (C-C motif) ligand 2 (CCL2) or thioglycollate, respectively. Our results reveal long-lasting changes to progenitor cells of monocytes/macrophages by a 2-hour dmPGE 2 pulse that, in turn, limits the migration of their daughter cells to chemoattractants and inflammatory mediators., (Copyright © 2017 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

A systemic immunosuppression follows UV irradiation of the skin of humans and mice. In this study, dendritic cells (DCs) differentiating from the bone marrow (BM) of UV-irradiated mice had a reduced ability to migrate toward the chemokine (C-C motif) ligand 21. Fewer DCs also accumulated in the peritoneal cavity of UV-chimeric mice (ie, mice transplanted with BM from UV-irradiated mice) after injection of an inflammatory stimulus into that site. We hypothesized that different metabolic states underpin altered DC motility. Compared with DCs from the BM of nonirradiated mice, those from UV-irradiated mice produced more lactate, consumed more glucose, and had greater glycolytic flux in a bioenergetics stress test. Greater expression of 3-hydroxyanthranilate 3,4-dioxygenase was identified as a potential contributor to increased glycolysis. Inhibition of 3-hydroxyanthranilate 3,4-dioxygenase by 6-chloro-dl-tryptophan prevented both increased lactate production and reduced migration toward chemokine (C-C motif) ligand 21 by DCs differentiated from BM of UV-irradiated mice. UV-induced prostaglandin E 2 has been implicated as an intermediary in the effects of UV radiation on BM cells. DCs differentiating from BM cells pulsed in vitro for 2 hours with dimethyl prostaglandin E 2 were functionally similar to those from the BM of UV-irradiated mice. Reduced migration of DCs to lymph nodes associated with increased glycolytic flux may contribute to their reduced ability to initiate new immune responses in UV-irradiated mice., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Impaired immune function has been implicated in the declining health and higher incidence of cancer in the elderly. However, age-related changes to immunity are not completely understood. Neutrophils and macrophages represent the first line of defence yet their ability to phagocytose pathogens decrease with aging. Cytotoxic T lymphocytes are critical in eliminating tumors, but T cell function is also compromised with aging. T cell responses can be regulated by macrophages and may depend on the functional phenotype macrophages adopt in response to microenvironmental signals. This can range from pro-inflammatory, anti-tumorigenic M1 to anti-inflammatory, pro-tumorigenic M2 macrophages. Macrophages in healthy elderly adipose and hepatic tissue exhibit a more pro-inflammatory M1 phenotype compared to young hosts whilst immunosuppressive M2 macrophages increase in elderly lymphoid tissues, lung and muscle. These M2-like macrophages demonstrate altered responses to stimuli. Recent studies suggest that neutrophils also regulate T cell function and, like macrophages, neutrophil function is modulated with aging. It is possible that age-modified tissue-specific macrophages and neutrophils contribute to chronic low-grade inflammation that is associated with dysregulated macrophage-mediated immunosuppression, which together are responsible for development of multiple pathologies, including cancer. This review discusses recent advances in macrophage and neutrophil biology in healthy aging and cancer., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Macrophages interact with cells in every organ to facilitate tissue development, function and repair. However, the close interaction between macrophages and parenchymal cells can be subverted in disease, particularly cancer. Motility is an essential capacity for macrophages to be able to carry out their various roles. In cancers, the macrophage's interstitial migratory ability is frequently co-opted by tumor cells to enable escape from the primary tumor and metastatic spread. Macrophage accumulation within and movement through a tumor is often stimulated by tumor cell production of the mononuclear phagocytic growth factor, colony-stimulating factor-1 (CSF-1). CSF-1 also regulates macrophage survival, proliferation and differentiation, and its many effects are transduced by its receptor, the CSF-1R, via phosphotyrosine motif-activated signals. Mutational analysis of CSF-1R signaling indicates that the major mediators of CSF-1-induced motility are phosphatidyl-inositol-3 kinase (PI3K) and one or more Src family kinase (SFK), which activate signals to adhesion, actin polymerization, polarization and, ultimately, migration and invasion in macrophages. The macrophage transcriptome, including that of the motility machinery, is very complex and highly responsive to the environment, with selective expression of proteins and splice variants rarely found in other cell types. Thus, their unique motility machinery can be specifically targeted to block macrophage migration, and thereby, inhibit tumor invasion and metastasis., Competing Interests: The authors declare no conflict of interest.

Macrophages infiltrate cancers and promote progression to invasion and metastasis. To directly examine tumor-associated macrophages (TAMs) and tumor cells interacting and co-migrating in a three-dimensional (3D) environment, we have developed a co-culture model that uses a PyVmT mouse mammary tumor-derived cell line and mouse bone marrow-derived macrophages (BMM). The Py8119 cell line was cloned from a spontaneous mammary tumor in a Tg(MMTV:LTR-PyVmT) C57Bl/6 mouse and these cells form 3-dimensional (3D) spheroids under conditions of low adhesion. Co-cultured BMM infiltrate the Py8119 mammospheres and embedding of the infiltrated mammospheres in Matrigel leads to subsequent invasion of both cell types into the surrounding matrix. This physiologically relevant co-culture model enables examination of two critical steps in the promotion of invasion and metastasis by BMM: 1) macrophage infiltration into the mammosphere and, 2) subsequent invasion of macrophages and tumor cells into the matrix. Our methodology allows for quantification of BMM infiltration rates into Py8119 mammospheres and demonstrates that subsequent tumor cell invasion is dependent upon the presence of infiltrated macrophages. This method is also effective for screening macrophage motility inhibitors. Thus, we have developed a robust 3D in vitro co-culture assay that demonstrates a central role for macrophage motility in the promotion of tumor cell invasion., Competing Interests: The authors have declared that no competing interests exist.

A major role of colony-stimulating factor-1 is to stimulate the differentiation of mononuclear phagocytic lineage cells into adherent, motile, mature macrophages. The colony-stimulating factor-1 receptor transduces colony-stimulating factor-1 signaling, and we have shown previously that phosphatidylinositol 3-kinase p110δ is a critical mediator of colony-stimulating factor-1-stimulated motility through the colony-stimulating factor-1 receptor pY721 motif. Src family kinases are also implicated in the regulation of macrophage motility and in colony-stimulating factor-1 receptor signaling, although functional redundancy of the multiple SFKs expressed in macrophages makes it challenging to delineate their specific functions. We report a comprehensive analysis of individual Src family kinase expression in macrophage cell lines and primary macrophages and demonstrate colony-stimulating factor-1-induced changes in Src family kinase subcellular localization, which provides clues to their distinct and redundant functions in macrophages. Moreover, expression of individual Src family kinases is both species specific and dependent on colony-stimulating factor-1-induced macrophage differentiation. Hck associated with the activated colony-stimulating factor-1 receptor, whereas Lyn associated with the receptor in a constitutive manner. Consistent with this, inhibitor studies revealed that Src family kinases were important for both colony-stimulating factor-1 receptor activation and colony-stimulating factor-1-induced macrophage spreading, motility, and invasion. Distinct colony-stimulating factor-1-induced changes in the subcellular localization of individual SFKs suggest specific roles for these Src family kinases in the macrophage response to colony-stimulating factor-1., (© Society for Leukocyte Biology.)

Colony-stimulating factor-1 (CSF-1), also known as macrophage-colony stimulating factor (M-CSF), is the primary growth factor regulating survival, proliferation and differentiation of macrophages. It is also a potent chemokine for macrophages and monocytes. Signaling via the CSF-1 receptor (CSF-1R) is necessary for the production of almost all tissue resident macrophage populations and these macrophages participate, via trophic mechanisms, in the normal development and homeostasis of tissues and organs in which they reside, including the mammary gland. The drawback of this close interaction between macrophages and parenchymal cells is that dysregulation of macrophage trophic functions assists in the development and progression of many cancers, including breast cancer. Furthermore, tumour cells secrete CSF-1 to attract more macrophages to the tumour microenvironment where CSF-1R signaling frequently drives the behaviour of these tumour-associated macrophages (TAMs) to promote tumour progression and metastasis. Evidence is mounting that treated tumours secrete more CSF-1 and the increased recruitment of TAMs limits treatment efficacy. Thus, therapeutic targeting of the CSF-1R to inhibit TAM function is likely to enhance tumour response and improve patient outcomes in the treatment of cancer, including breast cancer.

Colony stimulating factor-1 (CSF-1) stimulates mononuclear phagocytic cell survival, growth and differentiation into macrophages through activation and autophosphorylation of the CSF-1 receptor (CSF-1R). We have previously demonstrated that CSF-1-induced phosphorylation of Y721 (pY721) in the receptor kinase insert triggers its association with the p85 regulatory subunit of phosphoinositide 3'-kinase (PI3K). Binding of p85 PI3K to the CSF-1R pY721 motif activates the associated p110 PI3K catalytic subunit and stimulates spreading and motility in macrophages and enhancement of tumor cell invasion. Here we show that pY721-based signaling is necessary for CSF-1-stimulated PtdIns(3,4,5)P production. While primary bone marrow-derived macrophages and the immortalized bone marrow-derived macrophage cell line M-/-.WT express all three class IA PI3K isoforms, p110δ predominates in the cell line. Treatment with p110δ-specific inhibitors demonstrates that the hematopoietically enriched isoform, p110δ, mediates CSF-1-regulated spreading and invasion in macrophages. Thus GS-1101, a potent and selective p110δ inhibitor, may have therapeutic potential by targeting the infiltrative capacity of tumor-associated macrophages that is critical for their enhancement of tumor invasion and metastasis., (© 2013 FEBS.)

Colony-stimulating factor-1 (CSF-1)-stimulated CSF-1 receptor (CSF-1R) tyrosine phosphorylation initiates survival, proliferation, and differentiation signaling pathways in macrophages. Either activation loop Y807F or juxtamembrane domain (JMD) Y559F mutations severely compromise CSF-1-regulated proliferation and differentiation. YEF, a CSF-1R in which all eight tyrosines phosphorylated in the activated receptor were mutated to phenylalanine, lacks in vitro kinase activity and in vivo CSF-1-regulated tyrosine phosphorylation. The addition of Tyr-807 alone to the YEF backbone (Y807AB) led to CSF-1-independent but receptor kinase-dependent proliferation, without detectable activation loop Tyr-807 phosphorylation. The addition of Tyr-559 alone (Y559AB) supported a low level of CSF-1-independent proliferation that was slightly enhanced by CSF-1, indicating that Tyr-559 has a positive Tyr-807-independent effect. Consistent with the postulated autoinhibitory role of the JMD Tyr-559 and its relief by ligand-induced Tyr-559 phosphorylation, the addition of Tyr-559 to the Y807AB background suppressed proliferation in the absence of CSF-1, but restored most of the CSF-1-stimulated proliferation. Full restoration of kinase activation and proliferation required the additional add back of JMD Tyr-544. Inhibitor experiments indicate that the constitutive proliferation of Y807AB macrophages is mediated by the phosphatidylinositol 3-kinase (PI3K) and ERK1/2 pathways, whereas proliferation of WT and Y559,807AB macrophages is, in addition, contributed to by Src family kinase (SFK)-dependent pathways. Thus Tyr-807 confers sufficient kinase activity for strong CSF-1-independent proliferation, whereas Tyr-559 maintains the receptor in an inactive state. Tyr-559 phosphorylation releases this restraint and may also contribute to the CSF-1-regulated proliferative response by activating Src family kinase.

Colony stimulating factor-1 (CSF-1, also known as macrophage-colony stimulating factor, M-CSF) has long been known as the primary growth factor regulating survival, proliferation and differentiation of macrophages and other mononuclear phagocytic (MNP) lineage cells. CSF-1 was subsequently identified as a monocyte/macrophage chemokine, a capacity now recognized to be integral to many of the deleterious as well as positive roles of macrophages in development, homeostasis and disease. The pleiotrophic actions of CSF-1 are all transduced by its high affinity receptor, the CSF-1R, a receptor tyrosine kinase (RTK) and the cellular homologue of the v-fms oncoprotein. While the CSF-1R is the sole receptor for CSF-1, an alternative functional ligand for the receptor, interleukin-34 (IL-34), was recently identified. CSF-1-induced CSF-1R activation triggers autophosphorylation of several intracellular tyrosine residues, leading to initiation of an array of phosphotyrosine-based signaling cascades that mediate the wide variety of cellular responses to CSF-1. Dissecting the contributions of the different phosphorylated tyrosine motifs of the receptor to downstream signaling events in macrophages is not only important for our understanding of CSF-1R function, but also for the development of inhibitors to treat diseases where infiltrating macrophages contribute to their progression. This review will outline our current understanding of the CSF-1/CSF-1R signaling axis and describe how a novel macrophage cell line system, which allows examination of CSF-1R signaling in a mature macrophage context, is helping us to tease apart the diverse signaling pathways initiated by CSF-1R activation.

Macrophages are terminally differentiated cells of the mononuclear phagocytic lineage and develop under the stimulus of their primary growth and differentiation factor, CSF-1. Although they differentiate into heterogeneous populations, depending upon their tissue of residence, motility is an important aspect of their function. To facilitate their migration through tissues, macrophages express a unique range of adhesion and cytoskeletal proteins. Notably, macrophages do not form large, stable adhesions or actin stress fibers but rely on small, short lived point contacts, focal complexes and podosomes for traction. Thus, macrophages are built to respond rapidly to migratory stimuli. As well as triggering growth and differentiation, CSF-1 is also a chemokine that regulates macrophage migration via activation the CSF-1 receptor tyrosine kinase. CSF-1R autophosphorylation of several intracellular tyrosine residues leads to association and activation of many downstream signaling molecules. However, phosphorylation of just one residue, Y721, mediates association of PI3K with the receptor to activate the major motility signaling pathways in macrophages. Dissection of these pathways will identify drug targets for the inhibition of diseases in which macrophages contribute to adverse outcomes.

Colony stimulating factor-1 (CSF-1) regulates macrophage morphology and motility, as well as mononuclear phagocytic cell proliferation and differentiation. The CSF-1 receptor (CSF-1R) transduces these pleiotropic signals through autophosphorylation of eight intracellular tyrosine residues. We have used a novel bone-marrow-derived macrophage cell line system to examine specific signaling pathways activated by tyrosine-phosphorylated CSF-1R in macrophages. Screening of macrophages expressing a single species of CSF-1R with individual tyrosine-to-phenylalanine residue mutations revealed striking morphological alterations upon mutation of Y721. M⁻/⁻.Y721F cells were apolar and ruffled poorly in response to CSF-1. Y721-P-mediated CSF-1R signaling regulated adhesion and actin polymerization to control macrophage spreading and motility. Moreover, the reduced motility of M⁻/⁻.Y721F macrophages was associated with their reduced capacity to enhance carcinoma cell invasion. Y721 phosphorylation mediated the direct association of the p85 subunit of phosphoinositide 3-kinase (PI3K) with the CSF-1R, but not that of phospholipase C (PLC) γ2, and induced polarized PtdIns(3,4,5)P₃ production at the putative leading edge, implicating PI3K as a major regulator of CSF-1-induced macrophage motility. The Y721-P-motif-based motility signaling was at least partially independent of both Akt and increased Rac and Cdc42 activation but mediated the rapid and transient association of an unidentified ~170 kDa phosphorylated protein with either Rac-GTP or Cdc42-GTP. These studies identify CSF-1R-Y721-P-PI3K signaling as a major pathway in CSF-1-regulated macrophage motility and provide a starting point for the discovery of the immediate downstream signaling events.

CSF-1 is the major regulator of tissue macrophage development and function. A GM-CSF-dependent, CSF-1 receptor (CSF-1R)-deficient F4/80(hi)Mac-1(+)Gr1(-)CD11c(+) bone marrow macrophage (BMM) line (MacCsf1r-/-) was developed to study the roles of the eight intracellular CSF-1R tyrosines phosphorylated upon receptor activation. Retroviral expression of the wild-type CSF-1R rescued the CSF-1-induced survival, proliferation, differentiation, and morphological characteristics of primary BMM. Mutation of all eight tyrosines failed to rescue, whereas the individual Y --> F mutants (544, 559, 697, 706, 721, 807, 921, 974) rescued these CSF-1-inducible phenotypes to varying degrees. The juxtamembrane domain Y559F and activation loop Y807F mutations severely compromised proliferation and differentiation, whereas Y706, Y721F, and Y974F mutations altered morphological responses, and Y706F increased differentiation. Despite their retention of significant in vitro tyrosine kinase activity, Y559F and Y807F mutants exhibited severely impaired in vivo receptor tyrosine phosphorylation, consistent with the existence of cellular mechanisms inhibiting CSF-1R tyrosine phosphorylation that are relieved by phosphorylation of these two sites. The MacCsf1r-/- macrophage line will facilitate genetic and proteomic approaches to CSF-1R structure/function studies in the major disease-related CSF-1R-expressing cell type.

Macrophages are a key component of the innate immune system. In this study, we investigate how focal adhesion kinase (FAK) and the related kinase Pyk2 integrate adhesion signaling and growth factor receptor signaling to regulate diverse macrophage functions. Primary bone marrow macrophages isolated from mice in which FAK is conditionally deleted from cells of the myeloid lineage exhibited elevated protrusive activity, altered adhesion dynamics, impaired chemotaxis, elevated basal Rac1 activity, and a marked inability to form stable lamellipodia necessary for directional locomotion. The contribution of FAK to macrophage function in vitro was substantiated in vivo by the finding that recruitment of monocytes to sites of inflammation was impaired in the absence of FAK. Decreased Pyk2 expression in primary macrophages also resulted in a diminution of invasive capacity. However, the combined loss of FAK and Pyk2 had no greater effect than the loss of either molecule alone, indicating that both kinases function within the same pathway to promote invasion.

We describe three siblings with holoprosencephaly, recurrent infections, and increased peripheral blood monocytes. These children were born to apparently healthy parents in a family with one unaffected child. Affected individuals had microcephaly, severe developmental delay, failure to thrive, and brachydactyly. The clinical courses were complicated by endocrine dysfunction, multiple respiratory, and skin infections. Laboratory studies showed normal karyotypes, normal lymphocyte function, and a peripheral blood monocytosis with markedly abnormal morphology. Mutation analysis of the seven genes (SHH, ZIC2, SIX3, TGI, FTDGF1, GLI2, and PTCH) known to be involved in holoprosencephaly was normal. This is the first report demonstrating an association between abnormal mononuclear phagocytes and holoprosencephaly., ((c) 2006 Wiley-Liss, Inc.)

Previous studies have shown that macrophages and tumor cells are comigratory in mammary tumors and that these cell types are mutually dependent for invasion. Here we show that macrophages and tumor cells are necessary and sufficient for comigration and invasion into collagen I and that this process involves a paracrine loop. Macrophages express epidermal growth factor (EGF), which promotes the formation of elongated protrusions and cell invasion by carcinoma cells. Colony stimulating factor 1 (CSF-1) produced by carcinoma cells promotes the expression of EGF by macrophages. In addition, EGF promotes the expression of CSF-1 by carcinoma cells thereby generating a positive feedback loop. Disruption of this loop by blockade of either EGF receptor or CSF-1 receptor signaling is sufficient to inhibit both macrophage and tumor cell migration and invasion.

Macrophage actin-associated tyrosine phosphorylated protein (MAYP) belongs to the Pombe Cdc15 homology (PCH) family of proteins involved in the regulation of actin-based functions including cell adhesion and motility. In mouse macrophages, MAYP is tyrosine phosphorylated after activation of the colony-stimulating factor-1 receptor (CSF-1R), which also induces actin reorganization, membrane ruffling, cell spreading, polarization, and migration. Because MAYP associates with F-actin, we investigated the function of MAYP in regulating actin organization in macrophages. Overexpression of MAYP decreased CSF-1-induced membrane ruffling and increased filopodia formation, motility and CSF-1-mediated chemotaxis. The opposite phenotype was observed with reduced expression of MAYP, indicating that MAYP is a negative regulator of CSF-1-induced membrane ruffling and positively regulates formation of filopodia and directional migration. Overexpression of MAYP led to a reduction in total macrophage F-actin content but was associated with increased actin bundling. Consistent with this, purified MAYP bundled F-actin and regulated its turnover in vitro. In addition, MAYP colocalized with cortical and filopodial F-actin in vivo. Because filopodia are postulated to increase directional motility by acting as environmental sensors, the MAYP-stimulated increase in directional movement may be at least partly explained by enhancement of filopodia formation.

BCL6 is a potent transcriptional repressor that plays important roles in germinal center formation, T helper cell differentiation and lymphomagenesis and regulates expression of several chemokine genes in macrophages. In a further investigation of its role in macrophages, we show that BCL6 inactivation in primary bone marrow-derived macrophages leads to decreased polarization, motility and cell spreading accompanied by an increase in peripheral focal complexes, anchored F-actin bundles and cortical F-actin density. These changes were associated with excess RhoA activation. C3 transferase inhibition of RhoA activity reverted the adhesion structure phenotype, which was not affected by Rho kinase inhibitors, suggesting that other downstream effectors of Rho maintain this Bcl6(-/-) phenotype. Excess RhoA activation in BCL6-deficient macrophages is associated with a decrease in the p120RasGAP (RASA1)-mediated translocation of p190RhoGAP (GRLF1) to active RhoA at the plasma membrane and a reduction in cell surface expression of the CSF1R that has been reported to recruit RasGAP to the plasma membrane. Reconstitution of BCL6 expression in Bcl6(-/-) macrophages results in complete reversion of the morphological phenotype and a significant increase in cell surface CSF1R expression whereas overexpression of the CSF1R corrects the polarization and adhesion structure defects. These results demonstrate that BCL6 suppresses RhoA activity, largely through upregulation of surface CSF1R expression, to modulate cytoskeletal and adhesion structures and increase the motility of macrophages.

The transcription repressor BCL-6 is known to play critical roles in B-cell lymphomagenesis, germinal center formation, and balanced Th1/Th2 differentiation. In macrophages, although BCL-6 has also been shown to regulate the expression of several chemokine genes, its function in other aspects of macrophage biology has not been studied. In addition, the precise role of BCL-6 in cell proliferation is poorly understood in general. Here we report that BCL-6(-/-) macrophages hyperproliferate due to an accelerated G(1)/S transition accompanied by increased cyclin D2 and c-myc and decreased expression of p27. Crucial to this enhanced proliferation is spontaneous interleukin 6 (IL-6) production and signal transducer and activator of transcription 3 (STAT3) activation in BCL-6(-/-) macrophages. In colony-forming assays, BCL- 6(-/-) bone marrow progenitor cells form spontaneous macrophage colonies that can be inhibited by anti-IL-6 antibodies. Gene expression studies demonstrate that BCL-6 binds to several sequence motifs scattered in the IL-6 locus and can repress IL-6 transcription both in 293T cells and in macrophages. In conclusion, our results indicate that BCL-6 negatively regulates proliferation of the monocytic/macrophage lineage by suppressing an autocrine IL-6/STAT3-mediated gene expression program. Our work also suggests that BCL-6 prevents abnormal Th2 differentiation by suppressing basal level IL-6 production in antigen-presenting cells (APCs).

Most tissue macrophages and osteoclasts are regulated by colony-stimulating factor-1 (CSF-1, also known as macrophage CSF). The effects of CSF-1 are mediated by the CSF-1 receptor tyrosine kinase (CSF-1R), through autophosphorylation of CSF-1R and the subsequent phosphorylation of downstream molecules. Triggering this phosphorylation cascade increases gene transcription and protein translation, and induces cytoskeletal remodeling by several signaling pathways, leading to the survival, proliferation and differentiation of target cells. CSF-1-regulated tissue macrophages are important for innate immunity and for tissue development and function. Because CSF-1 regulates the survival, proliferation and chemotaxis of macrophages and supports their activation, this factor is involved in the pathogenesis of several diseases.

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein, thereby promoting cell-cycle progression. Cyclin D1 is overexpressed in hematopoetic and epithelial malignancies correlating with poor prognosis and metastasis in several cancer types. Because tumor-associated macrophages have been shown to enhance malignant progression and metastasis, and cyclin D1-deficient mice are resistant to oncogene-induced malignancies, we investigated the function of cyclin D1-/- bone marrow-derived macrophages. Cyclin D1 deficiency increased focal complex formation at the site of substratum contact, and enhanced macrophage adhesion, yielding a flattened, circular morphology with reduced membrane ruffles. Migration in response to wounding, cytokine-mediated chemotaxis, and transendothelial cell migration of cyclin D1-/- bone marrow-derived macrophages were all substantially reduced. Thus, apart from proliferative and possible motility defects in the tumor cells themselves, the reduced motility and invasiveness of cyclin D1-/- tumor-associated macrophages may contribute to the tumor resistance of these mice.

Primary macrophages isolated from hck(-/-)fgr(-/-) mice display altered morphology and F-actin cytoskeletal structures and reduced migration. The ability of phorbol myristyl acetate (PMA), a protein kinase C activator that has been reported to increase macrophage spreading and carcinoma cell motility, to rescue these hck(-/-)fgr(-/-) defects was tested. Although PMA-treated wild-type and hck(-/-)fgr(-/-) macrophages exhibited a similar flattened, spread phenotype, PMA did not rescue the hck(-/-)fgr(-/-) macrophage migration defect. Instead, both PMA-treated wild type and hck(-/-)fgr(-/-) macrophages were defective in spontaneous and chemotactic migration and tyrosine phosphorylation of the Cbl protooncoprotein was decreased in both. Moreover, c-cbl(-/-) macrophages displayed the same impairment of motility as hck(-/-)fgr(-/-) macrophages and a similar morphology with less polarization and more dorsal ruffling than wild-type macrophages. As Hck and Fgr expression and activity were not decreased in c-cbl(-/-) macrophages, these results suggest that Cbl is likely to be an important downstream mediator of the Src family kinase-regulated macrophage motility pathway., (Copyright 2003 Wiley-Liss, Inc.)

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.

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.

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 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.

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.

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.

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.

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.