Cell therapy for full-thickness wounds: are fetal dermal cells a potential source?

The application of autologous dermal fibroblasts has been shown to improve burn wound healing. However, a major hurdle is the availability of sufficient healthy skin as a cell source. We investigated fetal dermal cells as an alternative source for cell-based therapy for skin regeneration. Human (hFF), porcine fetal (pFF) or autologous dermal fibroblasts (AF) were seeded in a collagen-elastin substitute (Novomaix, NVM), which was applied in combination with an autologous split thickness skin graft (STSG) to evaluate the effects of these cells on wound healing in a porcine excisional wound model. Transplantation of wounds with NVM+hFF showed an increased influx of inflammatory cells (e.g., neutrophils, macrophages, CD4(+) and CD8(+) lymphocytes) compared to STSG, acellular NVM (Acell-NVM) and NVM+AF at post-surgery days 7 and/or 14. Wounds treated with NVM+pFF presented only an increase in CD8(+) lymphocyte influx. Furthermore, reduced alpha-smooth muscle actin (αSMA) expression in wound areas and reduced contraction of the wounds was observed with NVM+AF compared to Acell-NVM. Xenogeneic transplantation of NVM+hFF increased αSMA expression in wounds compared to NVM+AF. An improved scar quality was observed for wounds treated with NVM+AF compared to Acell-NVM, NVM+hFF and NVM+pFF at day 56. In conclusion, application of autologous fibroblasts improved the overall outcome of wound healing in comparison to fetal dermal cells and Acell-NVM, whereas application of fetal dermal fibroblasts in NVM did not improve wound healing of full-thickness wounds in a porcine model. Although human fetal dermal cells demonstrated an increased immune response, this did not seem to affect scar quality.

Minimal extracorporeal circulation (MECC) does not result in less hypertrophic scar formation as compared to conventional extracorporeal circulation (CECC) with dexamethasone.

INTRODUCTION: Cardiopulmonary bypass surgery is associated with a systemic inflammatory response through the interaction of air, blood and synthetic components in the bypass system and the physical trauma of surgery. An alternative cardiopulmonary bypass system, minimal extracorporeal circulation (MECC), has shown promising results in terms of reducing the inflammatory response. We hypothesized that this system may reduce pathological excessive scarring. To study this assumption, the effects of MECC and the effects of conventional extracorporeal circulation (CECC) with dexamethasone on skin scarring were compared in a standardized wound-healing model. METHODS AND RESULTS: Pre-sternal scars were evaluated prospectively at four and 12 months postoperatively. The height and width of the scars were measured, using a slide caliper and sonography. The scars were scored using the validated Patient and Observer Scar Assessment Scale. Additional risk factors for hypertrophic scar formation were identified by means of a questionnaire. During surgery, MECC was used in 45 patients and CECC/dexamethasone in 42 patients. Four months postoperatively, 22 patients of the MECC group (49%) and 18 patients in the CECC/dexamethasone group (43%) had developed hypertrophic scars. Twelve months postoperatively, the hypertrophic scars in four patients of the MECC group and in two patients of the CECC/dexamethasone group had become normotrophic. In 18 patients of the MECC group (38%) and 16 patients of the CECC group (41%) the scars remained hypertrophic at 12 months. These differences between the two groups were not statistically significant. CONCLUSION: MECC does not reduce hypertrophic scar formation compared with CECC with dexamethasone, but its use is more beneficial than the use of CECC/dexamethasone because of the circulatory and immunological advantages and because treatment with dexamethasone can be omitted.

Reversal of rocuronium-induced neuromuscular block with sugammadex in heart failure patients: a prospective observational study.

The aim of this study was to assess the hemodynamic stability and efficacy of 2 mg/kg sugammadex in reversing rocuronium-induced neuromuscular block in patients with heart failure. Twelve patients who had an ejection fraction < or = 25% and who were undergoing general anesthesia for cardiac resynchronization therapy, an automated implantable cardioverter-defibrillator, or battery replacement of the device were included. Neuromuscular function was monitored by acceleromyography of the adductor pollicis muscle. Each patient received 0.6 mg/kg of rocuronium and maintenance doses of 0.1 mg/kg when required. When the second twitch appeared at the end of surgery, the patients received 2 mg/kg sugammadex. After the administration of sugammadex, the time for recovery to a normalized train-of-four (TOF) ratio of 0.9 was 2.78 +/- 0.67 min. Blood pressure and heart rate remained stable up to 10 min after the administration of sugammadex and then increased by the 30-min assessment. Three patients had episodes of SpO2 < 90% in the postanesthesia care unit. No sugammadex-related adverse events were reported. Sugammadex can adequately restore neuromuscular function in heart failure patients under hemodynamically stable conditions. However, longer reversal times are required than previously observed in healthy, young patients.

Angiogenesis in peritoneal dialysis.

Long-term exposure to peritoneal dialysis fluid induces morphological alterations, including angiogenesis, leading to a loss of ultrafiltration (UF) capacity. We discuss the effect of different factors in peritoneal dialysis (PD) on angiogenesis. In addition, we describe the process of angiogenesis and the possible role of different cell types in the peritoneum upon PD contributing to new blood vessel formation. Furthermore, we review several interventions used in our rat PD exposure model to decrease angiogenesis in PD. Moreover, we show new data on the use of sunitinib to inhibit angiogenesis in this rat model. Although various interventions seem to be promising, well-randomised clinical trials showing absolute prevention of angiogenesis and UF failure are, yet, still missing. To make real progress in PD treatment, the aim should be to prevent angiogenesis as well as peritoneal fibrosis and PD-induced inflammation.

Mycotic aneurysm of the extracranial carotid artery: a case report and review of the literature.

Aneurysm of the extracranial carotid artery is a rare condition; mycotic aneurysms are even less common. They always need surgical treatment to prevent possible rupture, embolisation or death. The literature before 1980 mentions ligation as the only treatment for mycotic aneurysms of the carotid artery, obviously with a high morbidity and mortality rate. We describe an 85-year-old male with a left carotid artery mycotic aneurysm. He presented with a history of Transient Ischaemic Attack (TIA) four weeks after a gastrointestinal infection. Resection of the aneurysm and interposition with autologous vein was performed. Escherichia coli was isolated from the excised tissue. Primary resection of the aneurysm with autologous vein interposition, in association with prolonged antibiotic therapy, is the preferred strategy. Prompt diagnosis and aggressive treatment is essential to prevent serious complications.

Ultrastructure of mononuclear phagocytes developing in liquid bone marrow cultures. A study on peroxidatic activity.

Monoblasts, promonocytes, and macrophages in in vitro cultures of murine bone marrow were studied ultrastructurally, with special attention to peroxidatic activity. Monoblasts show peroxidatic activity in the rough endoplasmic reticulum and nuclear envelope as well as in the granules. The presence of peroxidatic activity in the Golgi apparatus could not be determined. Promonocytes have peroxidase-positive rough endoplasmic reticulum, Golgi apparatus, nuclear envelope, and granules, as previously reported. During culture, cells are formed with peroxidatic activity similar to that of monocytes or exudate macrophages (positive granules; negative Golgi apparatus, RER, and nuclear envelope); we call these cells early macrophages. In addition, transitional macrophages with both positive granules and positive RER, nuclear envelope, negative Golgi apparatus (as in exudate- resident macrophages in vivo), and mature macrophages with peroxidatic activity only in the RER and nuclear envelope (as in resident macrophages in vivo) were found. A considerable number of cells without detectable peroxidatic activity were also encountered. Our finding that macrophages with the peroxidatic pattern of monocytes (early macrophages), exudate-resident macrophages (transitional macrophages), and resident macrophages (mature macrophages), develop in vitro from proliferating precursor cells deriving from the bone marrow, demonstrates once again that resident macrophages in tissues originate from precursor cells in the bone marrow. Therefore, this conclusion can no longer be challenged on the basis of a cytochemical difference between monocytes and exudate macrophages on the one hand and resident macrophages on the other.

The joint association of air pollution and noise from road traffic with cardiovascular mortality in a cohort study.

OBJECTIVES: Associations between cardiovascular mortality and air pollution and noise together were investigated. METHODS: Data from the ongoing Netherlands Cohort Study on Diet and Cancer (120,852 subjects; follow-up 1987-1996) were used. Cox proportional hazard analyses were conducted for the association between cardiovascular mortality and exposure to black smoke, traffic intensity on the nearest road and road traffic noise at the home address. RESULTS: The correlations between traffic noise and background black smoke, and traffic intensity on the nearest road were moderate at 0.24 and 0.30, respectively. Traffic intensity was associated with cardiovascular mortality, with highest relative risk (95% confidence interval) for ischaemic heart disease (IHD) mortality being 1.11 (1.03 to 1.20) (increment 10,000 motor vehicles/24 h). Relative risks for black smoke concentrations were elevated for cerebrovascular (1.39 (0.99 to 1.94)) and heart failure mortality (1.75 (1.00 to 3.05)) (increment 10 microg/m(3)). These associations were insensitive to adjustment for traffic noise. There was an excess of cardiovascular mortality in the highest noise category (>65 dB(A)), with elevated risks for IHD (1.15 (0.86 to 1.53)) and heart failure mortality (1.99 (1.05 to 3.79)). After adjustment for black smoke and traffic intensity, noise risk reduced to unity for IHD mortality and was slightly reduced for heart failure mortality. CONCLUSIONS: Associations between black smoke concentrations and traffic intensity on the nearest road with specific cardiovascular causes of death were not explained by traffic noise in this study.

Associations between traumatic stress symptoms, pain and bio-active components in burn wounds.

OBJECTIVE: Pain and traumatic stress symptoms often co-occur. Evidence suggests that the neuropeptide oxytocine and pro-inflammatory cytokines are associated with both stress and pain. The aim of this pilot study was to explore relations between self-reported pain and traumatic stress, oxytocin and three cytokines in burn wounds. METHODS: An observational study in three burn centres was performed. Patients were invited to participate in the study when deep dermal injury was suspected. Patients completed the Impact of Event Scale (IES), a self-report questionnaire assessing traumatic stress symptoms, and they rated their pain the day prior to surgery. During surgery, eschar (i.e., burned tissue) was collected and stored at -80 ° C until analysis. When the data collection was complete, oxytocin and cytokine levels were analysed. RESULTS: Eschar from 53 patients was collected. Pain and stress scores were available from 42 and 36 patients respectively. Spearman correlational analyses showed an association between lower oxytocin levels at wound site and a higher total IES score (r = -0.37) and pain (r = -0.32). Mann-Whitney U tests comparing groups scoring high or low on pain or stress confirmed these associations. CONCLUSION: These analyses lend support to a hormonal pathway that may explain how psychological distress affects pain at skin level in patients with traumatic stress symptoms.

Differences in peritoneal response after exposure to low-GDP bicarbonate/lactate-buffered dialysis solution compared to conventional dialysis solution in a uremic mouse model.

BACKGROUND: Long-term exposure of conventional peritoneal dialysis (PD) fluid is associated with structural membrane alterations and technique failure. Previously, it has been shown that infiltrating IL-17-secreting CD4+T cells and pro-fibrotic M2 macrophages play a critical role in the PD-induced pathogenesis. Although more biocompatible PD solutions are recognized to better preserve the peritoneal membrane integrity, the impact of these fluids on the composition of the peritoneal cell infiltrate is unknown. MATERIALS AND METHODS: In a uremic PD mouse model, we compared the effects of daily instillation of standard lactate (LS) or bicarbonate/lactate-buffered solutions (BLS) and respective controls on peritoneal fibrosis, vascularisation, and inflammation. RESULTS: Daily exposure of LS fluid during a period of 8 weeks resulted in a peritoneal increase of αSMA and collagen accompanied with new vessel formation compared to the BLS group. Effluent from LS-treated mouse showed a higher percentage of CD4+ IL-17+ cell population while BLS exposure resulted in an increased macrophage population. Significantly enhanced inflammatory cytokines such as TGFß1, TNFα, INFγ, and MIP-1ß were detected in the effluent of BLS-exposed mice when compared to other groups. Further, immunohistochemistry of macrophage subset infiltrates in the BLS group confirmed a higher ratio of pro-inflammatory M1 macrophages over the pro-fibrotic M2 subset compared to LS. CONCLUSION: Development of the peritoneal fibrosis and angiogenesis was prevented in the BLS-exposed mice, which may underlie its improved biocompatibility. Peritoneal recruitment of M1 macrophages and lower number of CD4+ IL-17+ cells might explain the peritoneal integrity preservation observed in BLS-exposed mouse.

The delicate balance of macrophages in colorectal cancer; their role in tumour development and therapeutic potential.

Most tumours are heavily infiltrated by immune cells. This has been correlated with either a good or a bad patient prognosis, depending on the (sub) type of immune cells. Macrophages represent one of the most prominent leukocyte populations in the majority of tumours. Functions of macrophages range from cytotoxicity, to stimulation of tumour growth by secretion of cytokines, growth and angiogenic factors, or suppressing immune responses. In most tumours macrophages are described as cells with immune suppressing, and wound healing properties, which aids tumour development. Yet, increasing evidence shows that macrophages are potent inhibitors of tumour growth in colorectal cancer. Macrophages in this respect show high plasticity. The presence of high macrophage numbers in the tumour may therefore become advantageous, if cells can be reprogrammed from tumour promoting macrophages into potent effector cells. Enhancing cytotoxic properties of macrophages by microbial products, pro-inflammatory cytokines or monoclonal antibody therapy are promising possibilities, and are currently tested in clinical trials.

In vitro cultured fetal fibroblasts have myofibroblast-associated characteristics and produce a fibrotic-like environment upon stimulation with TGF-ß1: Is there a thin line between fetal scarless healing and fibrosis?

Transforming growth factor-ß (TGF-ß) is a cytokine occurring in three isoforms with an important function in development and wound healing. In wound healing, prolonged TGF-ß signaling results in myofibroblast differentiation and fibrosis. In contrast, the developing second-trimester fetal skin contains high levels of all three TGF-ß isoforms but still has the intrinsic capacity to heal without scarring. Insight into TGF-ß signal transduction during fetal wound healing might lead to methods to control the signaling pathway during adult wound healing. In this study, we imitated wound healing in vitro by stimulating fibroblasts with TGF-ß1 and examining myofibroblast differentiation. The aim was to gain insight into TGF-ß signaling in human fibroblasts from fetal and adult dermis. First, TGF-ß1 stimulation resulted in similar or even more severe upregulation of myofibroblast-associated genes in fetal fibroblasts compared to adult fibroblasts. Second, fetal fibroblasts also had higher protein levels of myofibroblast-marker α-smooth muscle actin (α-SMA). Third, stimulated fetal fibroblasts in collagen matrices had higher protein levels of α-SMA, produced more of the fibrotic protein fibronectin splice-variant extra domain A (FnEDA), and showed enhanced contraction. Finally, fetal fibroblasts also produced significant higher levels of TGF-ß1. Altogether, these data show that in vitro cultured fetal fibroblasts have myofibroblast-associated characteristics and do produce a fibrotic environment. As healthy fetal skin has high levels of TGF-ß1, FnEDA, and collagen-III as well, these findings correlate with the in vivo situation. Therefore, our study demonstrates that there are similarities between fetal skin development and fibrosis and shows the necessity to discriminate between these processes.

Cell cycle specific effects of tumor necrosis factor alpha in monocyte mediated leukemic cell death and the role of beta 2-integrins.

Human monocytes are involved in host defense against neoplastic cells. In view of cellular immunotherapy with cytotoxic monocytes in minimal residual disease of acute myeloid leukemia we have studied the role of monocytes in cell cycle dependent leukemic cell death of U937, THP-1, and HL-60 cells in vitro. Leukemic cells separated in G1 of the cell cycle by countercurrent centrifugal elutriation were highly susceptible to monocyte mediated cytotoxicity, whereas cells in S and G2-M were less sensitive or completely resistant as compared to unfractionated control cells. HL-60 cells resistant to cytotoxic monocytes became sensitive to monocyte mediated cytotoxicity upon differentiation induction with 1,25-dihydroxyvitamin D3 which paralleled an accumulation of cells in G1 of the cell cycle. The differences in susceptibility of cell phase separated populations to monocyte mediated cytotoxicity paralleled differences in sensitivity to the cytotoxic effects of tumor necrosis factor alpha, as secreted by gamma-interferon activated monocytes. Furthermore, monocyte mediated cytotoxicity was markedly inhibited in the presence of anti-CD11/CD18 monoclonal antibodies recognizing the alpha and beta chains of the beta 2-integrin adhesion proteins. By fluorescence activated cell sorter immunofluorescence a marked increase in mean fluorescence density of the beta 2-integrins could be demonstrated on cells in G1 of the cell cycle as compared to unseparated leukemic cells. A decrease in mean fluorescence density was shown for cells in G2-M. By blocking experiments with anti-CD11/CD18 monoclonal antibodies, the differences in mean fluorescence density were functionally relevant since cells in G1 were shown to be the most sensitive cells to beta 2-integrin dependent monocyte mediated cytotoxicity. In conclusion these data show that differences in sensitivity to tumor necrosis factor and in the expression of beta 2-integrins may play a central role in cell cycle dependent monocyte mediated antileukemic activity.

Currently known risk factors for hypertrophic skin scarring: A review.

OBJECTIVE: The study aims to provide an overview of risk factors for hypertrophic scarring. BACKGROUND: Hypertrophic skin scarring remains a major concern in medicine and causes considerable morbidity. Despite extensive research on this topic, the precise mechanism of excessive scarring is still unknown. In addition, the current literature lacks an overview of the possible risk factors in the development of hypertrophic scars. METHODS: PubMed searches were performed on risk factors for hypertrophic scar (HTS) formation. RESULTS: Eleven studies suggesting nine factors associated with HTS formation were found. Studies concerning chemotherapy, age, stretch, infection, and smoking have a moderate to high strength of evidence, but some other factors have not been studied in a convincing manner or are still disputed. CONCLUSIONS: Risk factors for HTS formation are young age, bacterial colonization, and skin subjected to stretch. Chemotherapy, statins, and smoking seem to play a protective role in HTS formation.

Particulate matter air pollution components and risk for lung cancer.

BACKGROUND: Particulate matter (PM) air pollution is a human lung carcinogen; however, the components responsible have not been identified. We assessed the associations between PM components and lung cancer incidence. METHODS: We used data from 14 cohort studies in eight European countries. We geocoded baseline addresses and assessed air pollution with land-use regression models for eight elements (Cu, Fe, K, Ni, S, Si, V and Zn) in size fractions of PM2.5 and PM10. We used Cox regression models with adjustment for potential confounders for cohort-specific analyses and random effect models for meta-analysis. RESULTS: The 245,782 cohort members contributed 3,229,220 person-years at risk. During follow-up (mean, 13.1 years), 1878 incident cases of lung cancer were diagnosed. In the meta-analyses, elevated hazard ratios (HRs) for lung cancer were associated with all elements except V; none was statistically significant. In analyses restricted to participants who did not change residence during follow-up, statistically significant associations were found for PM2.5 Cu (HR, 1.25; 95% CI, 1.01-1.53 per 5 ng/m(3)), PM10 Zn (1.28; 1.02-1.59 per 20 ng/m(3)), PM10 S (1.58; 1.03-2.44 per 200 ng/m(3)), PM10 Ni (1.59; 1.12-2.26 per 2 ng/m(3)) and PM10 K (1.17; 1.02-1.33 per 100 ng/m(3)). In two-pollutant models, associations between PM10 and PM2.5 and lung cancer were largely explained by PM2.5 S. CONCLUSIONS: This study indicates that the association between PM in air pollution and lung cancer can be attributed to various PM components and sources. PM containing S and Ni might be particularly important.

Effect of fixation protocols on in situ detection of L-selectin ligands.

In situ binding of (chimeric) proteins to tissue sections is a widely used method to identify ligands and their localization. Many different protocols for the fixation of frozen tissue sections are used for in situ binding studies. We report the effects of different fixation protocols on the binding pattern observed using in situ binding of an L-selectin-IgM chimeric protein to both rat lymph node and kidney tissue sections. L-selectin is a C-type lectin, expressed on leukocytes and is involved in both lymphocyte homing and migration upon inflammation. We show that different in situ binding patterns in rat kidney are observed using different fixation protocols, including glutaraldehyde, methanol, formaldehyde and acetone fixation. The observed staining is specific, as it can be blocked in the presence of EGTA, an L-selectin blocking antibody or by ligand competition. Enzymatic pre-treatment of the tissue sections using sialidase, heparitinase I or chondroitinase ABC has differential effects on in situ binding depending on tissue type and fixation protocol. These data indicate that special attention should be paid in choosing a fixation protocol for in situ binding studies, especially when using lectins. This could prevent biologically relevant ligands remaining undetected or wrong conclusions being drawn based on the localization of observed binding.

A functional study on the migration of human monocytes to human leukemic cell lines and the role of monocyte chemoattractant protein-1.

In the present study the migration of human monocytes towards the supernatants of five different human myeloid leukemic cell lines, four different human lymphatic leukemic cell lines and blasts derived from three different patients with acute myeloid leukemia (AML) was studied and the role of monocyte chemoattractant protein (MCP)-1 was established with an ELISA assay. Large differences in migration of monocytes towards the leukemic cell supernatants were shown (variation of approximately 10 to 150% compared to positive control), but high amounts of monocyte migration was always restricted to myeloid leukemic cells (cell lines or patient blasts). MCP-1 turned out to play a major role in the migration, firstly since there was a direct correlation between the amount of migration and the concentration of MCP-1 in the supernatants, and secondly since the addition of anti-hMCP-1 was able to inhibit migration to background level in all cases. Cytotoxicity experiments with a MTT test using MCP-1-stimulated monocytes against two human myeloid leukemic cell lines showed no increase in cell death compared to unstimulated monocytes. It is concluded that monocyte migration towards leukemic cells is restricted to the myeloid lineage and is regulated by MCP-1, which is produced in different amounts by the leukemic cells. Besides, MCP-1 does not increase the direct toxic effects of monocytes on leukemic cells.

Maturation-dependent susceptibility to monocyte-mediated cytotoxicity in acute myeloid leukemia.

In view of cellular immunotherapy with cytotoxic monocytes in minimal residual leukemia we have studied the effects of monocytes on the growth and survival of leukemic cells from cell lines and from patients with acute myeloid leukemia (AML). Using highly purified and interferon-gamma (IFN gamma) activated human monocytes, monocyte-mediated cytotoxicity (MMC) was evaluated in an MTT-based colorimetric cytotoxicity assay against six human leukemic cell lines (U937, THP1, KG1, K562, HL60, and 1,25(OH)2D3 differentiated HL60 cells) and cells from AML patients. Leukemic cells from cell lines with an immature phenotype were found to be resistant to MMC, whereas leukemic cells with a more mature and monocytic phenotype were sensitive. This paralleled the sensitivity to tumor necrosis factor-alpha (TNF-alpha). AML cells from patients with an immature phenotype (FAB-M1/M5A) were significantly less sensitive to MMC as compared to more mature AML cells (FAB-M2/M4/M5B). The growth stimulatory effects of non-activated monocytes on immature AML cells could be abrogated in the presence of IFN gamma or IL-3 and GM-CSF. In addition, these cytokines further potentiated MMC, preferentially affecting cells with a more mature phenotype. AML cells with an immunologically immature phenotype (CD34(high), HLA-Dr(low), CD13(low), CD14(low)) were revealed as the least sensitive cells to MMC. The growth stimulatory effects of IL-3/GM-CSF with or without TNF-alpha on AML cells correlated with resistance to MMC. In addition, the cytolytic effects of TNF-alpha in the presence of IFN gamma correlated with an increased susceptibility of AML cells to MMC. In conclusion, our data strongly indicate that MMC is related to maturation in AML, which is correlated to the differential stimulatory and cytolytic effects of monocyte-derived cytokines such as IL-3, GM-CSF, and TNF-alpha.

Macrophage phenotype regulation by colony-stimulating factors at bone marrow level.

Macrophages (m phi s) can be divided into several subpopulations, which differ in phenotype, function, and localization patterns. However, little is known about the mechanisms that regulate this heterogeneity. We investigated whether m phi heterogeneity is regulated by colony-stimulating factors (CSFs) at the bone marrow level. By clonal expansion of bone marrow-derived precursor cells in the presence of CSF-1, granulocyte-macrophage CSF or multi-CSF (interleukin-3), phenotypic heterogeneity was observed between m phi colonies. Heterogeneity was found especially when different CSF culture conditions were used but also between m phi colonies derived under the same CSF culture condition. Our results illustrate that CSFs from the bone marrow hemopoietic microenvironment are important for the induction of phenotypic heterogeneity within the progeny of cloned m phi precursor cells during maturation and differentiation in vitro.

Expression of Ia antigen in colonies of bone-marrow-derived macrophages.

Cells in colonies of culture-derived mouse and rat bone marrow macrophages were examined for membrane Ia antigen to determine if the steady-state expression of the antigen was restricted to macrophages derived from a distinct population of progenitor cells. Multiple subcultures of macrophages derived from single soft-agar colonies were tested for Ia+ cells on three different days of culture. About one-half of the colonies gave rise to subcultures that never contained Ia+ cells, about 40% yielded subcultures that contained some Ia+ cells on at least 2 of the 3 assay days, and about 10% of the colonies produced subcultures that contained Ia-bearing cells on all 3 assay days. Thus, when cultures were assayed at any one time for their content of Ia-bearing cells, the results raised the possibility of phenotypically distinct subpopulations of progenitors. However, sequential analyses of the subcultures revealed the variable expression of Ia on cells from at least one-half of the colonies. We conclude that, under steady-state conditions, the presence of Ia antigen on bone marrow-derived macrophages is not clonally restricted. That a T-cell-derived lymphokine induced Ia antigen on essentially all the cells in most of the colonies of macrophages confirms their potential to express the antigen.

Heterogeneity of macrophages in the rat evidenced by variability in determinants: two new anti-rat macrophage antibodies against a heterodimer of 160 and 95 kd (CD11/CD18).

A set of three monoclonal antibodies (MoAbs), ED1, ED2, and ED3, has been shown to recognize in situ different subsets of macrophages in the rat. This macrophage diversity can be correlated with differences in stage of differentiation of cells belonging to one lineage. The present study quantifies this antigen distribution in the macrophage fractions of several lymphoid organs provided by Percoll centrifugation. Four new MoAbs (ED4, ED7, ED8, and ED9) raised against macrophages are included in this study. The tissue distribution of each of the four new MoAbs is determined by immuno- and enzyme-histochemistry on cryostat sections. The MoAbs recognize distinct subpopulations of macrophages. The new MoAbs ED4, ED7, ED8, and ED9 recognize granulocytes and other unrelated cell types, as well as cells of the mononuclear phagocyte system. ED7 and ED8 recognize a surface heterodimer of Mr 160,000 and 95,000.