Adult Peripheral Blood Monocytes Research Articles

Human CD36hi monocytes induce Foxp3+ CD25+ T cells with regulatory functions from CD4 and CD8 subsets.

The fetal and neonatal immune systems are uniquely poised to generate tolerance to self, maternal and environmental antigens encountered in the womb and shortly after birth. However, the tolerogenic nature of fetal and neonatal immunity can be detrimental in the context of pathogens, leading to overwhelming bacterial infections or chronic viral infections. A variety of mechanisms contribute to fetal and neonatal tolerance, including a propensity to generate Foxp3+ regulatory T cells (Treg cells). However, the mechanism(s) of fetal Foxp3+ T-cell differentiation, the specific antigen-presenting cells required and factors that inhibit Treg generation after the neonatal period are poorly understood. Here, we demonstrate that a subset of CD14+ monocytes expressing the scavenger molecule, CD36, can generate CD4+ and CD8+ T cells that coexpress Foxp3 and T-bet from both umbilical cord blood. These Foxp3+ T-bet+ T cells potently suppress T-cell proliferation and ameliorate xenogeneic graft-versus-host disease. CD14+ CD36+ monocytes provide known Treg-inducing signals: membrane-bound transforming growth factor-beta and retinoic acid. Unexpectedly, adult peripheral blood monocytes are also capable of inducing Foxp3+ T cells from both cord blood and adult peripheral naïve T cells. The induction of Foxp3+ T cells in umbilical cord blood by monocytes was inhibited by the lymphoid fraction of adult peripheral blood cells. These studies highlight a novel immunoregulatory role of monocytes and suggest that antigen presentation by CD36hi monocytes may contribute to the peripheral development of Foxp3+ T-bet+ T cells with regulatory functions in both neonates and adults.

Human CD141+ dendritic cells generated from adult peripheral blood monocytes

Human CD141+ dendritic cells (DCs), specialized for cross-presentation, have been extensively studied in the development of DC-based therapy against cancer. A series of attempts was made to generate CD141+ DCs from cord blood CD34+ hematopoietic progenitors to overcome the practical limitation of in vivo rareness. In the present study, we identified a culture system that generates high CD141+ DCs. After culture of CD14+ monocytes in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 8 days, CD141 was detected on cells that adhered to the bottom of the culture plate. The attached cells exhibited typical features of immature monocyte-derived DCs (moDCs), except for higher CD86 expression, more dendrites and higher granularity compared with those that did not attach. With 3 additional days of culture, increased CD141 expression on the cells was retained along with adhesion ability and partial expression of CLEC9A, a c-type lectin receptor. Furthermore, the cells exhibited effective uptake of dead cells. Interestingly, the attached moDCs differently responded to polyinosinic:polycytidylic acid (poly I:C) stimulation as well as a mixed lymphocyte reaction. Collectively, our findings show that human CD141+ DCs can be sufficiently generated from peripheral blood CD14+ monocytes, potentiating further investigation into generation of higher yields of cross-priming human DCs in vitro.

B7 Costimulation and Intracellular Indoleamine 2,3-Dioxygenase Expression in Umbilical Cord Blood and Adult Peripheral Blood

Alterations in the expression of B7 costimulatory molecules and their receptors, as well as differences in the tryptophan (TRP) catabolic pathway, may influence immunological reactivity of umbilical cord blood (UCB) compared with adult peripheral blood (APB) T lymphocytes. We determined the frequency of activated (CD11b+) monocytes expressing B7-1, B7-2, B7-H1, and B7-H2, and that of T cells and CD4+ T helper cells expressing CD28, cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death-1 receptor, and inducible costimulator of T cells in UCB and APB samples using flow cytometry. We also examined the intracellular expression of indoleamine 2,3-dioxygenase (IDO) applying flow cytometry and plasma levels of TRP, kynurenine (KYN), and kynurenic acid using high-performance liquid chromatography. The level of CTLA-4 expression on CD4 cells was higher in UCB compared with in APB, indicating that the possibility of CD28-mediated costimulation may be decreased. The level of the corresponding costimulator molecule, B7-2, was also elevated. Therefore, this inhibitory relation may function to a higher extent in UCB than in APB. The plasma KYN to TRP (K/T) ratio was 2-fold higher in UCB compared with APB. However, the capacity of UCB monocytes to produce IDO compared with APB monocytes was lower, and reverse signaling via B7-2 in UCB monocytes was found to be immature, which suggests that the observed increase in K/T ratio may be due to placental, rather than fetal, overexpression of IDO in competent cells. These factors may all contribute to the previously observed reduced reactivity of UCB T lymphocytes compared to APB T cells.

Neonatal monocytes express antiapoptotic pattern of Bcl-2 proteins and show diminished apoptosis upon infection with Escherichia coli.

Neonates show sustained inflammation after a bacterial infection, which is associated with inflammatory diseases like bronchopulmonary dysplasia or periventricular leucomalacia. Physiologically, inflammation is terminated early after the removal of the invading pathogens by phagocytosis-induced cell death (PICD) of immune effector cells. Earlier results showed reduced PICD in neonatal monocytes. The underlying molecular mechanisms are unknown. We hypothesize that the reduced PICD in neonatal monocytes is regulated through the proteins of the B-cell lymphoma 2 (Bcl-2) protein family. mRNA and protein expression of Bcl-2 family proteins in cord blood and adult peripheral blood monocytes infected with Escherichia coli were analyzed by quantitative real-time PCR and flow cytometry and cytochrome c release by fluorescence microscopy. mRNA expression of antiapopototic Bcl-xL was upregulated in cord blood monocytes (CBMO), whereas proapoptotic Bim tended to be higher in peripheral blood monocytes (PBMO). Upon infection, Bax was more strongly expressed in PBMO compared with CBMO. The pro/antiapoptotic balance was skewed toward survival in CBMO and apoptosis in PBMO. Cytochome c release into the cytosol was enhanced in PBMO compared with CBMO. Bcl-2 proteins are involved in reduced PICD in neonatal monocytes. These findings are another step toward the understanding of sustained inflammation in neonates.

Imidazoquinoline Toll-like receptor 8 agonists activate human newborn monocytes and dendritic cells through adenosine-refractory and caspase-1–dependent pathways

Newborns have frequent infections and manifest impaired vaccine responses, motivating a search for neonatal vaccine adjuvants. Alum is a neonatal adjuvant but might confer a T(H)2 bias. Toll-like receptor (TLR) agonists are candidate adjuvants, but human neonatal cord blood monocytes demonstrate impaired T(H)1-polarizing responses to many TLR agonists caused by plasma adenosine acting through cyclic AMP. TLR8 agonists, including imidazoquinolines (IMQs), such as the small synthetic 3M-002, induce adult-level TNF from neonatal monocytes, but the scope and mechanisms of IMQ-induced activation of neonatal monocytes and monocyte-derived dendritic cells (MoDCs) have not been reported. We sought to characterize IMQ-induced activation of neonatal monocytes and MoDCs. Neonatal cord and adult peripheral blood monocytes and MoDCs were cultured in autologous plasma; levels of alum- and TLR agonist-induced cytokines and costimulatory molecules were measured. TLR8 and inflammasome function were assayed by using small interfering RNA and Western blotting/caspase-1 inhibitory peptide, respectively. The ontogeny of TLR8 agonist-induced cytokine responses was defined in rhesus macaque whole blood exvivo. IMQs were more potent and effective than alum at inducing TNF and IL-1β from monocytes. 3M-002 induced robust TLR pathway transcriptome activation and T(H)1-polarizing cytokine production in neonatal and adult monocytes and MoDCs, signaling through TLR8 in an adenosine/cyclic AMP-refractory manner. Newborn MoDCs displayed impaired LPS/ATP-induced caspase-1-mediated IL-1β production but robust 3M-002-induced caspase-1-mediated inflammasome activation independent of exogenous ATP. TLR8 IMQs induced robust TNF and IL-1β in whole blood of rhesus macaques at birth and infancy. IMQ TLR8 agonists engage adenosine-refractory TLR8 and inflammasome pathways to induce robust monocyte and MoDC activation and represent promising neonatal adjuvants.

Glyceraldehyde-3-phosphate dehydrogenase is a reliable internal control in Western blot analysis of leukocyte subpopulations from children

To study differences in the development of immunity, leukocytes from cord blood are often compared with those from adult peripheral blood. Western blot analysis is a common method for detecting proteins. In this study, we investigated the reliability of using different housekeeping proteins (β-actin, β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) as internal controls for different leukocyte subpopulations from infants, children, and adults. Our results showed that the expression levels of β-actin and β-tubulin were much lower in cord blood leukocytes than in adult leukocytes, and this expression pattern persisted in children up to 3 years old. Further study revealed that the β-actin expression level in newborns was especially lower in CD14-positive monocytes. However, cord blood and adult peripheral blood monocytes had similar expression levels of β-actin messenger RNA (mRNA). Further experiments showed that posttranslational regulation was responsible for the low β-actin expression level in neonatal monocytes. Thus, researchers should carefully assess the appropriate use of housekeeping gene-encoded proteins as internal standards to normalize samples for comparisons of different leukocyte populations from subjects of different ages. In this study, we determined that GAPDH was a more reliable internal control than others in Western blot analysis for comparing the development of immunity among infants, children, and adults.

Multi-nucleated giant cell formation from human cord blood monocytesin vitro, in comparison with adult peripheral blood monocytes

Multi-nucleated giant cells (MGCs; Langhans-type cell), formed from macrophage fusion, are recognized as a hallmark histological feature in chronic inflammation. However, their precise pathological role is still poorly understood, especially for microorganism pathogens in the neonatal immune system, which are capable of surviving intracellularly in phagocytes. To conduct a partial evaluation of the monocyte function of neonates, we investigated the ability of human cord blood monocytes to form MGCs in vitro by stimulating various cytokines and comparing them with adult peripheral blood monocytes. Monocytes from cord blood and adult peripheral blood were isolated and cultured for 14 days with cytokines known to induce MGC in vitro. The fusion index in experiments with a combination of interleukin (IL)-4 and macrophage colony-stimulating factor (M-CSF) and a combination of IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly lower in cord blood than in adult blood monocytes (P = 0.0018 and P = 0.0141, respectively). The number of nuclei per MGC was significantly lower in cord blood than in adult blood monocytes in experiments with IL-4 alone, the combination of IL-4 and M-CSF, and the combination of IL-4 and GM-CSF (P < 0.0001). These results suggest the possibility that the susceptibility of newborns to mycobacterium infection is due partly to impaired MGC formation.

Enhancement of Umbilical Cord Blood Cell Hematopoiesis by Maitake Beta-Glucan Is Mediated by Granulocyte Colony-Stimulating Factor Production

Maitake beta-glucan (MBG) is an extract from the fruit body of the Grifola frondosa mushroom that is being widely used to treat cancer in Asia. We have previously reported that MBG enhances mouse bone marrow cell (BMC) hematopoiesis in vitro and protects BMC from doxorubicin (DOX) toxicity. In the current study, we investigated the ability of MBG to enhance hematopoiesis and to reduce the toxic effects of DOX on fresh human umbilical cord blood (CB) cells. MBG treatment significantly enhanced the colony formation unit (CFU) response of granulocytes-macrophages (CFU-GM response) over the whole dose range of 12.5 to 100 microg/ml (P < 0.05). The addition of MBG to DOX-treated CB cells significantly protected granulocyte-macrophage colony formation from the toxicity of DOX, which otherwise produced strong hematopoietic repression. MBG also partially replaced recombinant human granulocyte colony-stimulating factor (rhG-CSF), as shown by a significant augmentation of the CFU-GM response in the absence of rhG-CSF. We found that MBG induces granulocyte colony-stimulating factor (G-CSF) production in CB CD33+ monocytes, as detected by intracellular cytokine flow cytometric assessment. In contrast, we found that adult peripheral blood monocytes did not produce a significant G-CSF response to MBG, whereas both adult and CB monocytes produced G-CSF in response to lipopolysaccharide. These studies provide the first evidence that MBG induces hematopoietic stem cell proliferation and differentiation of CFU-GM in umbilical CB cells and acts directly to induce G-CSF.

Differential Protein Expression of Zinc Finger Proteins and Cell Cycle Regulators between Lipopolysaccharide (LPS)-Activated Cord Blood (CB) and Adult Peripheral Blood (APB) Dendritic Cells (DC) Derived from Monocytes: Implication of CB Versus APB DC Differential Innate and Adaptive Immunity.

LPS induces the maturation and activation of dendritic cells in part by inducing toll-like receptor 4 (TLR4) and subsequent activation of specific signal transduction pathways (Medzhitov, Nat Rev Immunol., 2001). We and others have identified differential CB-DC versus APB-DC immune functional responses (Bradley/Cairo, Human Immunol., 2005). We have recently identified differential gene expression patterns in CB versus APB monocytes (Jiang/Cairo, J. of Immunol., 2004) and DC (Jiang/Cairo, Blood/ASH, 2004). Significant different gene expression patterns have been identified including cell surface molecules, cytokines/chemokines, signaling molecules, immune regulators and transcription factors. The differential expression of some of these genes may in part be responsible for the functional differences of CB versus APB DC. We therefore sought to examine quantitative differential protein expression patterns between CB and APB DC using proteomic tool (isotope-coded affinity tagging (ICATTM) followed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS)). Briefly, Mo were purified from fresh CB or APB and cultured for 8 days with GM-CSF and IL-4 (immature DC (iDC)) and activated by LPS for mature DC (mDC). Equal amounts of protein (1 mg) obtained from total cell lysates were reduced and labeled with isotope by mixing 1 unit of light (control) and heavy (treated) cleavable ICAT reagent and subjected to avidin affinity chromatography. Offline fractions were collected, digested with trypsin and analyzed by automated reverse phase nanospray LC-MS/MS. The MS/MS acquired data was searched with SEQUEST against amino acid sequences in the UniProt protein database to identify specific proteins. Our preliminary data demonstrated approximately 25 proteins overexpressed and 51 proteins underexpressed (> or < 1.5 fold threshold, respective) in APB iDC versus CB iDC. Similarly, there was a wide range of overexpressed (45) and underexpressed (108) proteins in APB mDC versus CB mDC. Specifically, our results indicated several Zinc finger proteins (292, 221, 474, 578) were expressed higher in APB versus CB mDC (2 to 5 fold). Two Ras-related small G-proteins Rab-3C and Ran-BP2 demonstrated 1.4 and 1.3 fold higher expression respectively in APB versus CB mDC. We also identified that interleukin-4 precursor revealed a 7.7 fold higher expression levels in APB DC versus CB mDC. In contrast, significant lower protein expression levels were demonstrated in APB versus CB DC, particularly cell cycle regulators cyclin I (3 fold), cyclin G-associated kinase (2 fold), Rb-like protein 2 (4.35 fold), PKC theta (2 fold). The expression of zinc finger proteins has been implicated to be associated with monocyte differentiation (Yuan, J. of Cancer Res., 2001) and dendritic cell differentiation (Li, Neuron, 2004). Additionally, Rab and Ran, small G-proteins, have been shown to be important for cell morphology and motility (Schmidt/Hall, Ann. Rev. Cell &Devel. Biol.,1998). In summary, this study reveals for the first time the global proteomic differences between CB and APB DC and provides novel insights into their differential immune function between CB versus APB DC.

Selective developmental defects of cord blood antigen-presenting cell subsets

Defective antigen-presenting cell (APC) function has been hypothesized to contribute to increased infection susceptibility in newborns. We used multiparameter flow cytometry to characterize APC subsets in adult peripheral blood (APB) and cord blood (CB). APB had a higher proportion of CD11c+ dendritic cells (DC), whereas CB mainly contained CD123+ DC. APB was enriched in CD16+CD11c+ DC subset, whereas CD34+CD11c-CD123lo cells were prominent in CB. Lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production was dampened in myeloid DC and monocytes from CB, whereas IL-1α production was not different. The reduction in TNF-α response did not appear to result from reduced surface detection of LPS, because CD14, toll-like receptor (TLR)-4 and TLR-2 levels were not reduced in CB APC compared with APB cells. Also, there was no correlation between TLR-2 or TLR-4 levels and TNF-α production in myeloid DC and monocytes. CB monocytes had lower surface HLA-DR immediately ex vivo. Both APB and CB monocytes upregulated HLA-DR after incubation, but an additional LPS-induced increase in HLA-DR was suggested only in APB monocytes. APB monocytes also showed a greater LPS-induced increase in CD40 expression. Together, our data show significant, selective differences in circulating APC between neonates and adults.

Significant decreased lipopolysaccharide (LPS)-activated cytokine/receptor, chemokine, immunoregulatory, transcription, apoptotic, signaling and cell structure gene expression in cord blood (CB) compared to adult peripheral blood (APB) monocytes by oligonucleotide gene expression microarray: insight into differential apb versus CB monocyte structure and function

Umbilical cord blood hematopoiesis and cellular immunity are developmentally immature when compared with adult peripheral blood (Cairo et al, Blood 90:4465,1997). We and others have demonstrated that umbilical cord blood transplantation (UCBT) is associated with decreased (grade III-IV) aGVHD, but delayed immune reconstitution (Abu-Ghosh/Cairo et al, BMT 24:535,1999). Recently, the contribution of allograft monocytes to the induction of GVHD and/or immune tolerance has been demonstrated (Aranha et al, Haematologica, 87:219, 2002). Monocytes are critically important in the generation of cytokines, chemokines, immune mediators and the initiation of the cellular immune responses. We and others have previously demonstrated significant dysregulated cytokine gene expression and protein production and in vitro functional activities of activated CB versus APB MNC. In this study we compared, by oligonucleotide microarray, the differential gene expression profiles of basal and LPS-activated APB versus CB monocytes. Briefly, Mo were purified from fresh CB or APB (N = 5) and stimulated with LPS (10 μg/ml, 18 hours). mRNA was isolated, reverse transcripted to cDNA, labeled and hybridized to oligonucleotides (Affymetrix, U95A) (Huang et al, Science 294:870, 2001). Data was analyzed by Microarray Suite Version 5.0 (Affymetrix) and hierarchical clustering analysis was performed by GeneSpring 5.0 software (Silicon Genetics). Quantitative real time PCR (LightCycler-RNA amplification SYBR Green I kit, Roche Molecular Biochemicals) was used to examine selected genes to confirm expression levels. We demonstrated patterns of significant differential amplified gene expression in LPS-activated APB versus CB monocytes including cytokine (G-CSF, 14 fold), chemokine (MIP-1α, 5 fold), immunoregulatory (MHC DRB1, 5 fold), transcription factor (JunB, 4 fold), signal transduction (STAT4, 5 fold), apoptotic regulation (BAX, 5 fold) and cell structure (ladinin 1, 6 fold) among others. These results suggest that some of these differentially expressed genes may in part be responsible for the differences in the incidence and severity of aGVHD, immunoreconstitution and/or the ability to cross more disparate HLA barriers following UCBT. Moreover, these data provide insight into the molecular basis for differential cellular function and immune responses between activated CB and APB monocytes. Umbilical cord blood hematopoiesis and cellular immunity are developmentally immature when compared with adult peripheral blood (Cairo et al, Blood 90:4465,1997). We and others have demonstrated that umbilical cord blood transplantation (UCBT) is associated with decreased (grade III-IV) aGVHD, but delayed immune reconstitution (Abu-Ghosh/Cairo et al, BMT 24:535,1999). Recently, the contribution of allograft monocytes to the induction of GVHD and/or immune tolerance has been demonstrated (Aranha et al, Haematologica, 87:219, 2002). Monocytes are critically important in the generation of cytokines, chemokines, immune mediators and the initiation of the cellular immune responses. We and others have previously demonstrated significant dysregulated cytokine gene expression and protein production and in vitro functional activities of activated CB versus APB MNC. In this study we compared, by oligonucleotide microarray, the differential gene expression profiles of basal and LPS-activated APB versus CB monocytes. Briefly, Mo were purified from fresh CB or APB (N = 5) and stimulated with LPS (10 μg/ml, 18 hours). mRNA was isolated, reverse transcripted to cDNA, labeled and hybridized to oligonucleotides (Affymetrix, U95A) (Huang et al, Science 294:870, 2001). Data was analyzed by Microarray Suite Version 5.0 (Affymetrix) and hierarchical clustering analysis was performed by GeneSpring 5.0 software (Silicon Genetics). Quantitative real time PCR (LightCycler-RNA amplification SYBR Green I kit, Roche Molecular Biochemicals) was used to examine selected genes to confirm expression levels. We demonstrated patterns of significant differential amplified gene expression in LPS-activated APB versus CB monocytes including cytokine (G-CSF, 14 fold), chemokine (MIP-1α, 5 fold), immunoregulatory (MHC DRB1, 5 fold), transcription factor (JunB, 4 fold), signal transduction (STAT4, 5 fold), apoptotic regulation (BAX, 5 fold) and cell structure (ladinin 1, 6 fold) among others. These results suggest that some of these differentially expressed genes may in part be responsible for the differences in the incidence and severity of aGVHD, immunoreconstitution and/or the ability to cross more disparate HLA barriers following UCBT. Moreover, these data provide insight into the molecular basis for differential cellular function and immune responses between activated CB and APB monocytes.

Changes of CD14 and CD1a expression in response to IL-4 and granulocyte-macrophage colony-stimulating factor are different in cord blood and adult blood monocytes.

Neonates are relatively immature in their immune response; thus, to further clarify the differences of monocyte function and differentiation between neonates and adults, we investigated their CD14(+)CD4(+) and CD14(+)CD16(+) monocyte subpopulations, production of IL-1beta and tumor necrosis factor-alpha induced by lipopolysaccharide, and their CD14 and CD1a phenotypic changes in response to IL-4 and granulocyte-macrophage colony-stimulating factor. Our results showed that 1) the expression of CD14 in cord blood monocytes was significantly lower than that in adult peripheral blood monocytes; 2) both the percentages of CD14(+)CD4(+) cells and CD14(+)CD16(+) cells among CD14(+) monocytes were also significantly lower in cord blood; 3) after stimulation by lipopolysaccharide for 72 h, production of both IL-1beta and tumor necrosis factor-alpha was lower in cord blood than that in adult peripheral blood; and 4) in response to IL-4 or GM-CSF, the phenotype development of CD14 and CD1a in cord blood and adult peripheral blood was different. Down-regulation of CD14 expression in response to IL-4 and GM-CSF was slower in cord blood monocytes than that in adult peripheral blood monocytes. After 9 d of culture in the presence of IL-4 and GM-CSF, the percentage of CD1a(+) monocytes was significantly more increased in cord blood than that in adult peripheral blood. The reduced expression of CD14 and other mature phenotype markers such as CD16 and CD4 as well as the reduced IL-1beta and tumor necrosis factor-alpha production may contribute to the impaired immune response of neonates. Slower down-regulation of CD14 by IL-4 and GM-CSF suggests that differential properties of cord blood monocytes in response to cellular stress signals take a longer time than those of adult peripheral blood monocytes.

Regulation of newborn and adult monocytes by PGE2: evidence of comparable differentiation status.

PGE2, which is produced in large amounts by monocytes, is considered to be an important autocrine factor in the regulation of the immune response. Altered sensitivity to PGE2 is known to reflect the differentiation status of monocytes/macrophages. Since newborn monocytes are thought to be less differentiated than adult peripheral blood monocytes, this study was designed to determine whether the diminished newborn monocyte function, relative to adult peripheral blood monocytes, might be the result of an altered autocrine responsiveness to PGE2. Phosphodiesterase activity as well as cAMP and TNF alpha levels in response to PGE2 were measured in newborn and adult monocytes, as an index of sensitivity to PGE2. Basal intracellular cAMP levels were reduced in cord monocytes compared with adult monocytes. Addition of increasing concentrations of PGE2 (10M(-10)-10M-6) caused similar fold increases in intracellular levels of cAMP for both populations. Lipopolysaccharide-induced TNF alpha production was significantly reduced in a similar fashion for both cord and adult monocytes at high (> or = 10(-7)M) PGE2 concentration. Phosphodiesterase activity was comparable in lysates from both populations. This study shows that PGE2 does not differentially influence newborn and adult monocyte responses and therefore that the diminished functional abilities of newborn monocytes is not due to altered autocrine responsiveness.

Diminished production of prostaglandin E2 by monocytes of newborns is due to altered fatty acid membrane content and reduced cyclooxygenase activity.

To determine the biochemical basis for reduced PGE2 expression by cord blood monocytes relative to adult peripheral blood monocytes, fatty acid composition of cell membranes together with the amount and activity of enzymes involved in the cyclooxygenase pathway were investigated. Gas chromatographic analysis of mononuclear cell fatty acids showed a significant reduction in long chain fatty acids, including arachidonic acid. Acylase and phospholipase A2 activity, together with lipocortin 1 levels, were similar in cord and adult monocytes. No difference in amounts of the 70-kDa cyclooxygenase type 1 enzyme was observed between the monocyte sources. The 74k-Da cyclooxygenase type 2 enzyme was synthesized de novo upon LPS stimulation in comparable amounts in both cord and adult monocytes, with both monocyte types responding with equal sensitivity to LPS. The ability of microsomes, prepared from cord monocytes, to convert arachidonic acid into eicosanoids of the cyclooxygenase pathway was reduced. In contrast, the ability of acetylated microsomes to convert PGG2 to PGE2 was comparable, suggesting that the deficiency in PGE2 production by cord monocytes is due to reduced activity, specifically at the cyclooxygenase-active site rather than the peroxidase active site. In conclusion, it appears that both reduced fatty acid content and diminished cyclooxygenase activity are responsible for diminished eicosanoid production by cord monocytes.

Membrane antigen and ligand receptor expression on neonatal monocytes.

The monocyte/macrophage cell lineage is an essential component of host defense. Functional deficiencies have been described in neonatal monocytes, but knowledge of membrane antigen and receptor ligand expression in neonatal monocytes is incomplete. In this study, antigen and receptor ligand expression of cord blood monocytes (CBM) was examined and compared to adult peripheral blood monocytes (PBM). Leu-M3 and Leu-M5 antigens were shown to be present on all CBM. Using dual fluorescence microfluorometry, the percentage and intensity of expression of HLA-DR, CD4 antigens, Fc gamma and IL-2 receptors (IL-2R) on Leu-M3+ and Leu-M5+ CBM were compared to PBM. A lower percentage of expression of HLA-DR+ (87 +/- 3% vs. 95 +/- 1%, p = 0.02) and FC gamma RII+ (96 +/- 1% vs. 99 +/- 0.2%, p = 0.04) was noted on CBM. CD4, FC gamma RI, and FC gamma RIII expression on CBM were comparable to PBM. LPS stimulation of CBM induced IL-2R expression and enhanced HLA-DR antigen expression as seen previously on PBM. These findings indicate that CBM are phenotypically comparable to adult PBM with deficiencies localized only to a few specific areas.

Mononuclear Phagocytic Cells in Human Milk: HLA-DR and FcγR Ligand Expression

The study of the cellular immune components of human milk is essential in the understanding of the role human milk may play in protecting the nursing infant against infection. We have investigated some phenotypic characteristics of breast milk macrophages (BMM) and have compared them to the characteristics of adult peripheral blood monocytes (PBM) by using dual parameter flow microfluorometry. Most BMM expressed the monocyte/macrophage markers Leu-M3 and Leu-M5. The latter marker was present in high density (bright) on BMM, but the density of expression of Leu-M3 was higher on PBM than on BMM [median fluorescence intensity (MFI) 409 +/- 105 versus 203 +/- 106, p = 0.02]. The percentage of BMM (98 +/- 2) that expressed the HLA-DR antigen did not differ significantly from PBM, but the density of expression was higher on BMM (MFI 318 +/- 56 versus 264 +/- 41, p = 0.03). The HLA-DR expression of BMM was further enhanced after incubation with interferon-gamma for 36 h; however, receptor for interleukin-2 could not be induced on BMM by this treatment. The expression of the three classes of Fc gamma R was lower on BMM than on PBM, in percentage (Fc gamma RI 56 +/- 23 versus 79 +/- 17%, p = 0.02), density of expression (Fc gamma RIII MFI 71 +/- 20 versus 153 +/- 73, p = 0.002), or both (Fc gamma RII 74 +/- 22% versus 94 +/- 12%, p = 0.02, and MFI 115 +/- 53 versus 202 +/- 59, p = 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)