Phagocytic defects--monocytes/macrophages.

Mononuclear phagocytes originate from stem cells in the bone marrow which differentiate from monoblasts into promonocytes, then into circulating blood monocytes. Subsequently the monocytes can develop into macrophages and reside in a variety of tissues. Mononuclear phagocytes have cell surface receptors for a variety of substances (e.g., IgG, complement components, fibronectin, and sugars) and are capable of secreting a number of mediators (enzymes, complement components, coagulation components, and monokines). The tissue macrophages adapt to their environment and express unique differentiated functions that are related to various anatomic sites and organs (e.g., Kupffer cells, pulmonary alveolar macrophages, osteoclasts, microglia). Macrophages have the capacity to become "activated" by both specific and nonspecific immunologic stimuli and the "activated" macrophage has enhanced functional capabilities (e.g., tumoricidal, microbicidal, phagocytosis, secretion of mediators). Abnormal monocyte/macrophage function may be acquired or may be due to genetic or developmental disorders. Because of their central role in host defense (in inflammatory responses, in antigen presentation, and in immunoregulatory networks), monocyte/macrophage dysfunction may result in one or more pathophysiologic consequences: defects in monocyte maturation, deficiencies in the clearance of physiologic substrates in lysosomal diseases (e.g., Gaucher's disease, mucopolysaccharidoses, osteopetrosis, metachromatic leukodystrophy), decreased synthesis and secretion of mediators (complement component deficiencies), defects in microbicidal activity (chronic granulomatous disease) and defects which are acquired following infection and during chemotherapy (e.g., acquired immune deficiency syndrome).

Susceptibility of Propionibacterium acnes to killing and degradation by human neutrophils and monocytes in vitro.

Propionibacterium acnes, the target of inflammation in acne, was tested for its sensitivity to the bactericidal and degradative functions of human polymorphonuclear leukocytes (PMN), monocytes, and their fractions. P. acnes strains were not killed by PMN under any conditions and were variably killed by monocytes in the presence of serum from acne patients. Control strains of Staphylococcus aureus and Micrococcus lysodeicticus were susceptible to both PMN and monocyte killing. P. acnes strains were also not killed by lysozyme, chymotrypsin, H2O2, human serum, PMN granule lysate, and PMN and monocyte cell lysates. The organism was sensitive to the bactericidal activity of myeloperoxidase in acid pH. In addition, P. acnes was shown to be relatively resistant to the degradative action of PMN and monocyte lysates, whereas M. lysodeicticus, S. aureus, and Staphylococcus epidermidis were all degraded to various degrees. The moieties that were liberated from P. acnes by PMN enzymes were predominantly low in molecular weight (1,000 to 25,000) and were consistent with cell wall fragments.

Resolution of pulmonary inflammation.

Mononuclear phagocytes are suggested to play a major orchestrating role in the resolution of inflammatory processes in the lung. Particular emphasis is placed on their participation in the progression from a lesion with neutrophils as the dominant infiltrating cell to one that contains macrophages, on the macrophage's role in removing neutrophils and cell debris, and on their promotion of repair mechanisms. It is suggested that monocytes must mature into macrophages before they are capable of active participation in the resolution of inflammation.

Production of growth factor activity for fibroblasts by human monocyte-derived macrophages.

Macrophages, derived from human monocytes by in vitro culture, released a growth factor for rabbit lung fibroblasts. The release of growth factor was increased following stimulation with both soluble (lipopolysaccharide and phorbol myristate acetate) and particulate (opsonized zymosan) substances. Production of the growth factor activity was dependent on the length of time the monocytes were in culture, the presence of serum during the period of monocyte maturation, and macrophage protein synthesis. The inability of serum-deprived monocytes to produce growth factor could be reversed by adding back serum. Eicosatetraynoic acid and dexamethasone but not indomethacin inhibited the production of growth factor suggesting that arachidonic acid metabolites other than prostaglandins may regulate growth factor production.

Functional and biochemical studies of multinucleated giant cells derived from the culture of human monocytes.

We compared phagocytic and metabolic activities of multinucleated giant cells (MGC) and macrophages derived from human monocytes after 9-14 d in culture. Phagocytosis of sheep erythrocytes (E) coated with IgG, of E coated with IgM and complement, and of Candida albicans was comparable in MGC and macrophages. The same percentage of ingested fungi was killed by MGC (24 +/- 4%) and macrophages (21 +/- 5%). Approximately 70% of MGC and macrophages exhibited superoxide-dependent reduction of nitroblue tetrazolium during stimulation. Ia antigen was present on approximately 75% of both cell types. Analysis of cell populations separated by nuclear fluorescence indicated that beta-glucosaminidase, acid phosphatase, and beta-glucuronidase activity per cell was higher in MGC, but specific activity of these enzymes was greater in macrophages. These results suggest that MGC have the capacity to function like macrophages in host defense against infection.

Human serum induces maturation of human monocytes in vitro. Changes in cytolytic activity, intracellular lysosomal enzymes, and nonspecific esterase activity.

The dependence of human monocyte maturation in vitro on autologous serum was examined. If autologous serum was present during the monocyte culture, cytolysis of K562 target cells increased, intracellular levels of three lysosomal enzymes increased, and the fluoride-inhibitable esterase staining of the monocytes changed into a fluoride-resistant esterase stain (characteristic of more mature extravascular mononuclear phagocytes). Monocytes cultured in the presence and in the absence of serum also assumed different shapes. All of these changes were dependent on the concentration of autologous serum present (0-10%) and the length of time the monocytes were in culture (0-7 days). Lack of development by monocytes cultured in the absence of serum was not due to a general loss of the ability of these cells to function, because phagocytosis of antibody-coated erythrocytes was not lost following 7 days in culture in the absence of serum.

Contact with specific surfaces stimulates the production of the second component of complement (C2) in human peripheral blood monocytes via a lymphocyte factor.

Whole mononuclear cells plated on surfaces coated with the polymer, poly (2-hydroxyethyl methacrylate) (poly-HEMA) produced significantly less C2 when compared to production by cells on tissue culture plastic dishes. The reduction in C2 production was dependent on the amount of poly-HEMA used to coat the dishes and was not due to nonspecific damage of the cells or effects of the poly-HEMA on the hemolytic activity of C2. T and B lymphocytes, but not monocytes, plated on tissue culture plastic produced a soluble factor that increased the production of C2 in freshly adherent monocytes. Lymphocytes plated on the poly-HEMA surface did not produce this soluble factor, which was termed surface-dependent factor (SDF). Whole mononuclear cells plated on poly-HEMA were able to respond to SDF by increasing C2 production by the same percentage as cells on the tissue culture plastic. This suggested that the primary basis for the decreased production of C2 by monocytes in the whole mononuclear cells plated on the poly-HEMA was decreased production of SDF by the lymphocytes. The effect of the poly-HEMA surface on C2 production was probably related to a generalized alteration in maturation of monocytes into macrophages, for SDF had the same type of effect on beta-glucosaminidase levels in monocytes as seen with C2, except that the magnitude of the effect was less. These studies suggest that interaction of lymphocytes with surfaces may modulate the function of the lymphocytes. In addition, interaction of lymphocytes with surfaces and the production of SDF in vivo may be responsible for enhancing maturation of monocytes in tissues.

Protein synthesis-dependent and protein synthesis-independent secretion of lysosomal hydrolases from rabbit and human macrophages.

Rabbit alveolar macrophages and human monocyte-derived macrophages released lysosomal enzymes in response to a variety of stimuli. The release of these enzyme appeared to be under the control of at least two distinct mechanisms. The first involved a rapid release of preformed granule constituents in response to a phagocytic load. This release reaction was concentration- and time-dependent, was not affected by the protein synthesis inhibitors cycloheximide and puromycin, and resulted in a concomitant loss in intracellular enzyme levels. The second mechanism involved a prolonged secretion in response to lower concentration of stimuli, which increased with time, and was inhibited by cycloheximide and puromycin. The secretion did not result in the loss of intracellular enzyme stores; rather an induction of enzyme was seen following such stimulation, which resulted in increases in the total concentration within the cultures. This protein synthesis-dependent secretion of acid hydrolases from human macrophages varied for each lysosomal hydrolase and each stimulus. beta-Glucosaminidase synthesis and secretion was induced by low-dose opsonized zymosan, by latex particles, and by formaldehyde-treated SRBC. However, only the last mentioned stimulus caused release of acid phosphates although all three particles induced synthesis of the enzyme. None of the stimuli at these concentrations (10:1 particle to cell ratio) caused the release of beta-glucoronidase, although the enzyme was releasable if higher stimulus concentrations were used. In addition the enzyme was not inducible under these conditions. It is concluded that each of the acid hydrolases studied may be under different control in the human macrophage and that the cells may respond in a qualitatively different way to different types of phagocytosable stimuli.

Development of functional complement receptors during in vitro maturation of human monocytes into macrophages.

The development of Fc and C3 receptor function was evaluated during differentiation of human peripheral blood monocytes into macrophages in an in vitro culture system. At the time of isolation, monocytes formed rosettes with C3-coated erythrocytes (EIgMC) but did nt internalize them, whereas IgG-coated erythrocytes (EIgG) were both bound and ingested. During the first 24 hr in culture, IgG-mediated phagocytosis and C3-mediated rosette formation declined. After 7 days of in vitro differentiation, monocyte-derived macrophages were capable of ingesting 2 to 4 times as many EIgG as uncultured monocytes. In addition, macrophages demonstrated a new function--the ability to ingest C3-coated erythrocytes, as well as to efficiently bind them. The time course of the reappearance of receptor functions varied between different individuals, but by day 7, the activity of Fc and C3 receptors was vigorous for all individuals tested. Monocytes underwent differentiation whether they were cultured on glass coverslips or on a plastic surface. However, macrophages demonstrated C3-mediated ingestion earlier when cultured on plastic. Culturing monocytes on the different surfaces did not affect the time course of the reappearance of Fc receptor function, but did have a small effect on the magnitude of the response. These experiments demonstrate for the first time, that a defined population of human phagocytic cells can acquire the ability to mediate ingestion through their C3 receptors.

Humoral and formed elements of blood modulate the response of peripheral blood monocytes. I. Plasma and serum inhibit and platelets enhance monocyte adherence.

Human and rabbit peripheral blood monocytes normally adhere to plastic tissue culture plates in vitro when they are suspended in Hanks' media. Increasing amounts of autologous serum or heat-inactivated plasma in the cell suspensions prevented the adherence of both monocytes and lymphocytes. The inhibitory effect of plasma was separated into three areas of activity by chromatography on Sephacryl S-200. The profile of inhibitory activity did not coincide with the protein elution profile, suggesting that inhibition was not a nonspecific protein effect. A layer of adherent platelets overcame the inhibitory effect of plasma on monocyte adherence. Platelets selectively increased monocyte as opposed to lymphocyte adherence and this was specific for platelets in that neither neutrophils nor fibroblasts could substitute for platelets. Both plasma and platelets acted directly on monocytes.

Distribution of endotoxin (lipopolysaccharide) in the tissues of lipopolysaccharide-responsive and -unresponsive mice.

We examined the distribution of bacterial lipopolysaccharide (LPS) in LPS-responsive (C3H/St) and LPS-unresponsive (C3H/HeJ) mice. The results reported here demonstrate that the rates of removal of an immunological or a toxic dose of LPS from the circulation are the same in both strains of mice. C3H/St spleens accumulated significantly more LPS than C3H/HeJ spleens after the intravenous injection of either an immunogenic or a toxic dose of LPS. There was also a greater amount of LPS associated with cells teased from C3H/St spleens compared to those from C3H/HeJ spleens. After a toxic dose of LPS, there was more LPS in C3H/St lymph nodes, adrenals, lungs, kidneys, and heart than in the corresponding C3H/HeJ tissues. The accumulation of more LPS in tissues from C3H/St mice compared to C3H/HeJ mice suggests that these tissues are involved in the pathophysiological and, ultimately, the toxic effects of LPS. The differential accumulation of LPS in the tissues of these two strains may be the reason for the decreased responses of C3H/HeJ mice to LPS.

The role of an activatable esterase in immune-dependent phagocytosis by human neutrophils.

Diisopropylphosphofluridate, cyclohexyl alkylphosphonofluoridates, and cyclohexyl phenylalkylphosphonofluoridates, which are potent, irreversible inactivators of serine esterases, inhibit the phagocytosis of opsonized sheep erythrocytes by human neutrophils. Two types of inhibition were observed: a) 'cell-dependent' inhibition, which is determined by measuring the ingestion of neutrophils pre treated with the esterase inhibitors and washed; and b) 'phagocytosis-dependent' inhibition, which is due to the presence of the inhibitors during phagocytosis. With the cyclohexyl alkylphosphonofluoridates, phagocytosis-dependent inhibition was always greater than cell-dependent inhibition. Cell-dependent inhibition was irreversible and dependent on the duration of the incubation of neutrophils with inhibitor. Both types of inhibition were dependent on the concentration of the inhibitor. Poorly or non-phosphorylating analogues of the cyclohexyl alkylphosphonofluoridates of DFP were not inhibitory; nor did fluoride, the hydrolysis product of these inhibitors, inhibit ingestion under either condition. In addition, neither method of treating the neutrophils resulted in a decrease in neutrophil viability. Furthermore, pretreating the EAC1423 with the inhibitors did not decrease ingestion. We conclude that cell-dependent inhibition is due to the inactivation of an esterase required for phagocytosis which is in or on the neutrophil in an active form, and thus is susceptible to inhibition by the esterase inactivators before contact of the neutrophil with the phagocytic stimulus. Phagocytosis-dependent inhibition is interpreted as being due to inactivation of an esterase required for phagocytosis which is normally in an inactive precursor proesterase form that is activated by the interaction of the neutrophil with the phagocytic stimulus. The distinctly different inhibition profiles of the active and activatable esterases indicate that they are two different activities.