Search

Mo2 is a membrane antigen whose expression is associated with human cells enriched for monocytes and macrophages. Anti-Mo2 monoclonal antibody in the presence of rabbit complement causes an inhibition of the proliferative response of human peripheral blood leukocytes (PBL) to soluble antigens, that can be restored by the addition of fresh monocytes. Fluorescence-activated cell sorting (FACS) of PBL on the basis of Mo2 expression results in a population of Mo2+ cells (morphologically monocytes) that is selectively active in phagocytosis and cytolysis of antibody-sensitized human erythrocytes, and a population of Mo2- cells (morphologically lymphoid cells) that is nonphagocytic but selectively active in natural killing. These experiments indicate that expression of Mo2 is a useful marker for cells possessing the morphological and functional properties of monocytes.

Studies were carried out to investigate the nature of the lymphocyte membrane receptor involved in the specific lysis of [51Cr]EL4 target cells by BALB/c cytotoxic peritoneal exudate lymphocytes (PEL) in vitro. In confirmation of earlier related studies, the cytolytic reaction is markedly inhibited by trypsin treatment (1 mg of trypsin/10(6) PEL/ml; 37 C for 30 min) of the PEL. Trypsinized PEL recover their cytolytic activity after 6 hr of incubation in trypsin-free medium at 37 C; recovery is suppressed at 4 C. Significantly, trypsinization of PEL inhibits their ability to adsorb to EL4 target monolayers, indicating that lymphocyte-target cell binding is a trypsinsensitive step of the cytolytic interaction. Specificity of binding for EL4 is restored upon recovery from trypsin treatment. In addition, trypsin treatment, under conditions sufficient to inhibit cytolysis, causes a drastic nonspecific deletion of iodinatable membrane species from the surface of PEL. Thus, a direct correlation between altered cytolytic function and membrane structure cannot be made. Interestingly, trypsinized PEL retain the capacity to kill EL4 to which they are agglutinated by concanavalin-A, suggesting that the lytic mechanism, as distinguished from the binding receptor, is still intact. Papain and pronase also have an inhibitory effect on cytolysis, while neuraminidase causes a significant augmentation of lymphocyte-mediated cytolysis.

Mo3e is a protein (p 50,80) that is expressed on the surface of human monocytic cells after exposure in vitro to soluble activating factors that include bacterial lipopolysaccharide, muramyl dipeptide, and phorbol myristate acetate (PMA). The surface expression of Mo3e may represent a cellular event that occurs in response to the formation of "secondary messengers" that include diacylglycerol, inositol trisphosphate, and calcium ions. This postulate is based on the stimulatory effect of agents that can mimic the activity of endogenous diacylglycerol (PMA and other biologically active phorbol compounds, mezerein, and L-alpha-1,2 dioctanoylglycerol) and inositol trisphosphate (ionomycin) on Mo3e expression by U-937 and HL-60 cells. The inhibitory effect of phospholipid-active calmodulin inhibitors (trifluoperazine, chlorpromazine, and dibucaine), calcium antagonists (nicardipine and TMB-8), and EGTA further support the involvement of phospholipid- and calcium-dependent protein kinase (protein kinase C) and calcium ions in the up-modulation of Mo3e surface expression.

Mo5 is a 94-kd protein antigen expressed by human peripheral blood monocytes, neutrophils, and by all bone marrow myeloperoxidase-positive myeloid precursors (promyelocytes, myelocytes, metamyelocytes, and bands). Mo5 is borne by the malignant cells of 74% of patients (N = 27) with acute monocytic leukemia (French-American-British [FAB] group M4, M5), and 50% of patients (N = 38) with acute granulocytic leukemia (FAB M1, M2, and M3). Nonmyeloid cells in peripheral blood and bone marrow are Mo5-negative. The surface expression of Mo5 by myeloid cells is modulated by several experimental conditions: Exposure of neutrophils to calcium ionophore (1 mumol/L, 37 degrees C, ten minutes) under conditions resulting in degranulation of specific granules produces a three- to fourfold increase in the plasma membrane density of Mo5 antigen. This suggests that, in neutrophils, there is an intracellular pool of Mo5 antigen, which may be associated with specific granules, and that granule-associated Mo5 is translocated to the plasma membrane upon degranulation. Conversely, incubation of monocytes, neutrophils, U-937, and Mo5-positive leukemia cells in medium containing anti-Mo5 monoclonal antibody results in a significant decrease in surface Mo5 expression. This loss of surface Mo5 is a rapid, temperature-dependent process (occurring within 30 minutes at 37 degrees C) that is produced by divalent anti-Mo5 immunoglobulin [F(ab')2 but not F(ab)]. After down-modulation, Mo5 is reexpressed by monocytes within 48 hours. Mo5 is therefore a human myelomonocytic differentiation antigen whose expression is modulated up or down depending on the nature of extracellular stimuli.

Considerable information has been accumulated about the functional and metabolic differences that exist among human mononuclear phagocytes isolated from different anatomic sites. This heterogeneity may reflect the influence of environmental factors that stimulate circulating monocytes to differentiate in a distinctive manner, or may suggest the existence of predetermined subsets of macrophage precursors. The present study was designed to evaluate macrophage diversity at the membrane level by defining the expression of antigenic determinants on the surface of these cells. The expression of two new membrane differentiation antigens, BMM1 and PAM1, and of Mo1 and Mo2 were evaluated on blood monocytes, macrophages derived from cultured monocytes, breast milk macrophages (BMM), pulmonary alveolar macrophages (PAM), and peritoneal macrophages. Anti-BMM1 is a mouse IgG2a monoclonal antibody obtained by immunization with breast milk cells that recognizes an antigen expressed only on BMM but not on blood monocytes (or any other circulating cells), PAM, and peritoneal macrophages. BMM1 expression is also seen on HL60 promyelocytes induced toward macrophage differentiation by lymphokines but not by phorbol diester. Anti-PAM1, an IgG1 monoclonal antibody raised by immunization with PAM, reacts with an antigen found only on PAM. Blood monocytes, BMM, and peritoneal macrophages all share the previously described differentiation antigens, Mo1 and Mo2 (as well as Ia), whereas these markers are not found on PAM. BMM1 is associated with a two subunit surface protein of 46 and 40 Kd, and PAM1 resides on a 200 Kd polypeptide. The appearance of new antigens during differentiation (BMM1 on milk macrophages and PAM1 on PAM) and the disappearance of antigens more common to the macrophage lineage (absence of Mo1 and Mo2 on alveolar macrophages) indicate a significant heterogeneity at the membrane level among phagocytes obtained from different anatomic sites (but arising from a common myeloid precursor). The functional relevance of these structures selectively expressed by BMM and PAM is the object of further investigation.

The surface antigen phenotype of 30 patients with the blast phase of chronic myeloid leukemia (CML) was determined using a panel of monoclonal antibodies recognizing differentiation antigens of normal myeloid, erythroid, megakaryocyte, and lymphoid cells. Ten patients' cells expressed a phenotype corresponding to an immature myeloid cell and were felt to have "myeloid" blast crisis. None of these myeloid leukemias were TdT+ or responded to vincristine (V) and prednisone (P). Eleven patients expressed a phenotype similar to acute lymphoblastic leukemia cells and probably reflect maturation to an early B lymphocyte. All of these "lymphoid" leukemias were TdT+, and 67% of evaluable patients had a complete response to V and P. One leukemia had the phenotype of an erythroleukemia, one patient's cells expressed the phenotype of megakaryoblastic leukemia, and one leukemia had populations of both myeloid and lymphoid blasts. Six leukemias did not express surface markers characteristic of any lineage and were termed "undifferentiated." This group was heterogeneous with respect to TdT expression, but no patient had a complete response to V and P. Determination of surface antigen phenotype in CML blast crisis thus provides clinically useful information for the structuring of treatment protocols.

Mo1 alpha (formerly gp 110) is a neutrophil glycoprotein whose deficiency is associated with abnormalities in several neutrophil functions, including defects in adherence, chemotaxis, and phagocytosis. Examination of whole cells and subcellular components by the use of both immunological and electrophoretic techniques demonstrated that Mo1 alpha was located primarily in the specific granules but that a small portion was present in the plasma membrane, where it is exposed to the extracellular environment and can bind to anti-Mo1 antibody. During degranulation, Mo1 alpha is translocated from the specific granules to the plasma membrane, resulting in a 5-10-fold increase in the surface expression of this glycoprotein. These findings plus previous work suggest that plasma membrane-associated Mo1 alpha is needed for a normal interaction between neutrophils and underlying surfaces, and raise the possibility that the increase in surface adhesiveness of neutrophils that have discharged their specific granules might be due in part to the increase in the amount of Mo1 alpha in the plasma membranes of these degranulated cells.

Granulocytes from patients genetically deficient in the leukocyte glycoprotein family, Mo1, LFA-1, and Leu-M5 (P150,94), have defective complement receptor type III (CR3) activity as well as abnormal adhesion-dependent functions such as spreading, chemotaxis, and phagocytosis. To determine the contribution of the Mo1 heterodimer deficiency to the various functional aberrations observed in deficient granulocytes, we mapped the functional domains of Mo1 using several monoclonal antibodies to this molecule. In addition to iC3b binding, two granulocyte adhesion functions were examined: Cell spreading on plastic coverslips and chemotaxis. One monoclonal antibody to Mo1, 44, inhibits all three functions. Other monoclonal antibodies (903, Leu-15, and OKM10) inhibit iC3b binding to granulocytes but have no effect on cell spreading and/or chemotaxis. Another antibody, 904, has no significant inhibition of iC3b binding but inhibits spreading on plastic and chemotaxis. These studies suggest the presence of two functional domains in Mo1: one involved in iC3b binding and the other in enhancing certain granulocyte adhesion-dependent functions.

Mo1, 2, 3, and 4, and Plt-1 are a series of five distinct antigens detected on the surface of human peripheral blood monocytes by mouse monoclonal antibodies. Mo2 and 3 are restricted to the monocyte-macrophage series, while Mo1, as previously reported, is also expressed by human granulocytes and null cells. Mo3, as distinguished from Mo1 and Mo2, is weakly expressed by virgin peripheral blood monocytes but becomes well expressed if monocytes are cultured overnight at 37 degrees C. Mo4 is coexpressed by monocytes and platelets, while Plt-1 appears to be a platelet-specific antigen whose detection on monocytes reflects adherence of platelets to monocyte membranes. That Mo2-4 are true monocyte antigens is demonstrated by their resynthesis following protease treatment of monocytes (Mol expression is resistant to proteolytic digestion). During myeloid-monocyte differentiation, the Mo antigens are infrequently expressed by immature myeloid cells but are found at higher frequency on leukemic monocytic forms. Macrophages from cultured peripheral blood monocytes and HL-60 cells exposed to lymphokines or phorbol diester express Mo1-4, but noncirculating peritoneal macrophages lack Mo3. The Mo antigens are differentiation markers whose expression reflects membrane heterogeneity during myeloid-monocyte-macrophage maturation.

Previous studies using conventional hetero- or isoantisera have indicated the involvement of class II (Ia) molecules in presentation of soluble antigen by monocytes to inducer T lymphocytes, stimulation of inducer T cells in MLR, and recognition of Ia-bearing target cells by cytotoxic T lymphocytes (CTL). The experience in using monoclonal anti-Ia reagents capable of blocking these phenomena in the human system is limited. Recently, however, we have characterized a lytic IgG2a monoclonal antibody, 9-49, that binds to functionally significant class II molecules. This antibody blocks (in the absence of complement): (1) specific binding of peripheral blood lymphocytes (PBL) to antigen-pulsed monocyte monolayers, (2) proliferation of PBL in response to soluble antigen (tetanus toxoid or mumps) or cell surface class II antigen stimulation in allogeneic or autologous MLR, (3) proliferation of cloned T4+ (inducer) lymphocyte cell lines to class II antigens, (4) generation of cytotoxic lymphocytes during allogeneic MLR, and (5) recognition (and killing) of class II-bearing target cells by T4+ CTL clones. Proliferation and CTL activity of a T8+ clone is unaffected by the 9-49 antibody. These results indicate the usefulness of this monoclonal reagent in studies evaluating the functional role of Ia molecules in immune recognition phenomena.

Two antigens (Mo1 and Mo2) present on human peripheral blood monocytes have been defined by lytic IgM monoclonal antibodies. Both antigens are present on greater than 70% of adherent mononuclear cells (predominantly monocytes). Mo1 is expressed by monocytes, granulocytes, and Null cells, but is absent from T and B lymphocytes. Mo2, on the other hand, appears specific for peripheral blood monocytes. Neither antigen is present on Ia-positive B cell lines or on tumor cells from patients with B cell lymphoproliferative malignancies, further excluding the possibility that Mo1 and Mo2 are Ia antigens. Mo1 and Mo2 are, however, present on a significant number of blast cells from patients with monocytic leukemia (both myelomonocytic and pure monocytic variants), but relatively infrequently expressed by cells from patients with acute granulocytic leukemia. These results indicate that Mo1 and Mo2 are unique antigens that may represent distinct stages of late monocyte-granulocyte differentiation.

PAM1 is a 200-kDa polypeptide antigen present on lavaged human alveolar macrophages but not on monocytes, peritoneal macrophages, breast milk macrophages, or other normal hematopoietic cells studied by flow cytometry. We have characterized the distribution of expression of this antigen by cells in tissues by using immunohistologic techniques. Normal and diseased lung as well as lymph nodes, spleen, kidney, liver, GI tract, and skin were studied. PAM1 was expressed strongly on the surface and weakly in the cytoplasm of most alveolar macrophages in all 15 of the lung specimens. Occasional interstitial macrophages had weak to moderate staining for this antigen but the majority did not stain. The distribution, pattern, and intensity of staining for PAM1 was the same in normal lung specimens and those with interstitial pneumonitis, despite the increase in mononuclear cells in the latter. Dermal histiocytes and Kuppfer cells expressed PAM1 weakly. Sinus histiocytes in lymph nodes were moderately to strongly positive. Although lymphoid cell suspensions (tonsil) were negative by flow cytometry, five of six lymph nodes had positive cells by immunohistology. PAM1 was also detected on endothelial cells of splenic sinusoids in all 6 specimens but not on any other endothelium. Hence, while PAM1 is expressed most strongly on alveolar macrophages, it can also be demonstrated in other locations using sensitive immunohistologic techniques. Since circulating monocytes are antigen negative and some lung interstitial macrophages bear antigen, PAM1 may be a useful marker for studies of the differentiation of mononuclear cells in the lung.

Recent evidence suggests that the adenine compounds ATP, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), and adenosine have important regulatory effects on O2- responses of human neutrophils stimulated with the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP). Because of evidence that receptors on neutrophils may be modified by granule fusion events, we assessed the extent to which these adenine compounds affect fMLP and CR3 (C3bi) receptors on neutrophils and whether cytoplasmic granules are required for the ability of the adenine compounds to modify O2- responses in neutrophils stimulated with fMLP. Incubation of neutrophils with ATP gamma S or adenosine led to a decrease in numbers of fMLP receptors (17 and 9.2%, respectively) but no change in receptor affinity (Kd). Paradoxically, ATP gamma S caused an increase in CR3 receptors (Mo1, CD-11b antigen), suggesting that fMLP and CR3 receptors may be under separate control. The ability of the adenine compounds to modify O2- responses in fMLP-stimulated cells was equivalent in both neutrophils and cytoplasts, suggesting that the regulatory effect of ATP, ATP gamma S, and adenosine do not require the presence of cytoplasmic granules. ATP gamma S caused enhancement of O2- responses of neutrophils to phorbol 12-myristate 13-acetate, raising the possibility that ATP gamma S may be affecting late events in the signal transduction pathway.

Mo1 and Mo2 are membrane differentiation antigens expressed by human monocytes, and in the case of Mo1, by granulocytes and null cells. Mo1 antigen expression is resistant to protease treatment of intact monocytes while Mo2 is protease sensitive; Mo2 antigenic activity is regenerated by trypsin-treated monocytes during culture under conditions that favor protein synthesis. Further biochemical analysis reveals that Mo1 is associated with a glycoprotein consisting of two subunits of 94,000 and 155,000 MW. Mo2 resides on a glycoprotein of 55,000 MW. Immunoprecipitation of Mo1- and Mo2-bearing structures from lysates of internally labeled cells indicates that these proteins are synthesized by human monocytes.

A monoclonal antibody (904) that binds to a leukocyte cell adhesion-promoting glycoprotein, (Mo1; CD11b/CD18) was administered (1 mg/kg, iv.) to open chest anesthetized dogs 45 min after the induction of regional myocardial ischemia. Ischemia was produced by occluding the left circumflex coronary artery (LCX) for 90 min and then reperfusing for 6 h. There was no difference between control and antibody treated groups with respect to arterial blood pressure, heart rate, or LCX blood flow. Administration of antibody produced no observable effect on circulating neutrophil counts, suggesting that antibody-bound neutrophils were not cleared from the circulation. The mean size of myocardial infarct expressed as percentage of the area at risk of infarction that resulted was reduced by 46% with anti-Mo1 treatment (25.8 +/- 4.7%, n = 8) compared to control (47.6 +/- 5.7%, n = 8; P less than 0.01). The area at risk of infarction was similar between groups. Circulating (serum) antibody excess was confirmed in all 8 anti-Mo1 treated dogs by immunofluorescence analysis. Analysis of ST segment elevation on the electrocardiogram as an indicator of the severity of ischemia suggests that the anti-Mo1 reduces infarct size independent of the severity of ischemia. An additional group of dogs (n = 5) was tested with a control monoclonal antibody of the same subtype (murine IgG1) and was found to produce no significant reduction in myocardial infarct size. Accumulation of neutrophils within the myocardium was significantly attenuated with 904 treatment when analyzed by histological methods. These data demonstrate that administration of anti-Mo1 monoclonal antibody after the induction of regional myocardial ischemia results in reduced myocardial reperfusion injury as measured by ultimate infarct size.

To determine if inhibition of leukocyte adhesion and aggregation could improve postischemic ventricular dysfunction ("stunning"), a monoclonal antibody (904) that binds to the adhesion-promoting Mo1 glycoprotein on the cell surface of leukocytes was administered intravenously (0.5 mg/kg) to open-chest dogs before a 15-minute coronary occlusion. Ultrasonic crystals placed in ischemic and control myocardium were used to measure systolic wall thickening during a 15-minute occlusion of the left anterior descending artery and for 3 hours after reperfusion. Myocardial blood flow was measured with tracer-labeled microspheres before occlusion, after 10 minutes of occlusion, 3 minutes of reperfusion, and at 1 and 3 hours after reperfusion. Six animals receiving anti-Mo1 antibody had antibody excess demonstrated with immunofluorescence techniques at 5 minutes and 3 hours of reperfusion; this finding indicated saturation of binding sites. Five animals served as controls and received an antibody (murine immunoglobulin G) that does not influence neutrophils. The two groups did not differ hemodynamically during ischemia and reperfusion. Risk areas and myocardial blood flow were also not significantly different between the two groups. The main parameter used to define regional myocardial stunning, percentage systolic wall thickening in the ischemic/reperfused area, did not differ significantly between the two groups. Specimens from nonischemic myocardium were compared with ischemic specimens for myeloperoxidase content. There were no significant differences within or between groups. These data indicate that the anti-Mo1 monoclonal antibody (904) is not effective in improving the profound myocardial dysfunction that persists for 3 hours of reperfusion after 15 minutes of ischemia.

Mo3e is a protease-sensitive membrane antigen (p75,50) selectively expressed by human monocytic cells (monocytes and U-937 cells) stimulated in vitro by exposure to a variety of activating factors, including phorbol diester compounds, bacterial lipopolysaccharide (LPS), and muramyl dipeptide (MDP)(R.F. Todd et al., J. Immunol. 135, 3869, 1985). Here we report that primary and multiply-passaged cultures of HUVEC also express the Mo3e determinant after stimulation by phorbol myristate acetate (PMA) and related inducers of protein kinase C. As measured in a radioimmunoassay of anti-Mo3e antibody binding to monolayer cultures of HUVEC, unstimulated cells bore little if any Mo3e. After culture for 4-120 hr in medium containing PMA, 4 beta-phorbol dibutyrate, 4 beta-phorbol didecanoate, or mezerein (each at a concentration of 81 nM), or 1-oleoyl-2-acetoyl-sn-3-glycerol (1 mM), HUVEC were found to selectively express the Mo3e determinant. The magnitude of expression was dependent upon the concentration of the stimulus, maximal by 24 hr, and inhibited by cycloheximide. The combination of PMA and the calcium ionophore, ionomycin, had an additive or synergistic effect on HUVEC Mo3e expression. The biologically inactive phorbol compounds 4 beta-phorbol and 4 alpha-phorbol didecanoate failed to stimulate Mo3e expression. Also inactive as inducers of HUVEC Mo3e expression were crude lymphokine and monokine supernatants, recombinant human lymphokines (interferon-gamma and interleukin-2), recombinant human monokines (interleukin-1 and tumor necrosis factor), bacterial cell wall products including LPS and MDP, pharmacologic agents that increase intracellular cyclic adenosine monophosphate (prostaglandin E2, cholera toxin, theophylline, isoproterenol and isobutylmethylxanthine), lectins (Con A and PHA), and heparin. These results indicate that Mo3e is an inducible plasma membrane antigen of not only mononuclear phagocytes but also cultured HUVEC.

Events that lead to phagocytosis of complement (C3)- or IgG-coated particles after their interaction with specific cell surface receptors are poorly understood. Two mouse monoclonal antibodies (an IgM and an IgG2a) to a human granulocyte-monocyte surface membrane differentiation antigen (Mol) inhibited ingestion by granulocytes both of oil Red O particles opsonized with normal human serum or with IgG and of sheep erythrocytes sensitized with IgG. In addition, they specifically inhibited rosetting between phagocytes and sheep erythrocytes coated with C3bi, a fragment of the complement component C3, generated by cleaving C3b with C3b inactivator and beta IH protein. These monoclonal anti-Mol antibodies did not inhibit IgG Fc, C3b or C3d receptor-mediated binding of erythrocytes coated with the respective proteins. The Fab fragment of the IgG2a monoclonal antibody inhibited noncytotoxic enzyme release from granulocytes when these cells were stimulated with zymosan coated with C3bi. Electrophoretic transfer of polymorphonuclear leukocyte detergent lysates to nitrocellulose, followed by immunofixation with monoclonal antibody, showed that these antibodies were directed to a 155,000-mol wt glycoprotein. This surface membrane structure appears to be involved in Fc and C3 receptor-dependent phagocytosis and closely associated with the C3bi receptor.

Important insights into leukocyte differentiation and the cellular origins of leukemia have been gained through the use of monoclonal antibodies that define cell surface antigens and molecular probes that identify immunoglobulin and T cell receptor genes. Results of these studies have been combined with markers such as surface membrane and cytoplasmic immunoglobulin on B lymphocytes, sheep erythrocyte receptors on T lymphocytes, and cytochemical stains. Using all of the above markers, it is now clear that acute lymphoblastic leukemia (ALL) is heterogeneous. Although monoclonal antibodies are useful in distinguishing acute myeloid from acute lymphoid leukemias, they have less certain usefulness in the subclassification of acute myelogenous leukemia (AML). Attempts to subclassify AML by differentiation-associated antigens rather than by the French-American-British (FAB) classification are underway in order to document the potential prognostic usefulness of surface markers.

Mo1, a glycoprotein heterodimer (gp 155,95) that functions as an adhesion promoting molecule and as the C3bi receptor of human myeloid cells, is expressed in increased amounts in the plasma membrane after exposure of polymorphonuclear leukocytes (PMNs) to various stimuli. Previous studies have suggested that secondary granules represent an intracellular pool of Mo1 that, upon degranulation, fuse with the plasma membrane resulting in a tenfold increase in surface expression of Mo1. To determine the intracellular location of Mo1, we monitored Mo1 expression by immunofluorescence and compared it to the release of myeloperoxidase (MPO, a marker for the primary granules), vitamin B12 binding protein (B12BP, secondary granules), and gelatinase (gelatinase-containing organelles) following exposure to various stimuli. Human neutrophils stimulated with 20 mmol/L fluoride for 16 minutes exhibited a twofold increase in Mo1 expression and gelatinase release but no enhanced release of primary or secondary granular contents. In a similar fashion, incubation of cells at 37 degrees C for five minutes with 7.5 X 10(-9) to 10(-6) mol/L N-formyl-methionyl-leucyl-phenylalanine (FMLP) resulted in significant increases in both surface Mo1 expression (three- to fivefold) and gelatinase release (five- to eightfold) without significant release of either MPO or B12BP. In addition, both the fluoride and FMLP experiments demonstrated that Mo1 up-modulation alone is not sufficient to activate superoxide (O2-) production. These data indicate that at least one intracellular storage pool of Mo1 is the gelatinase-containing organelles and that their fusion with the plasma membrane results in increased expression of Mo1 on the cell surface.

A case is reported of hypercalcemia, prostatic adenocarcinoma and sarcoidosis. This represents the second reported occurrence of sarcoidosis and prostatic carcinoma, and illustrates the intriguing association between systemic sarcoidosis and malignancy. The reported experience demonstrating the association between sarcoidosis and malignant disease is reviewed.

The widespread assumption that cytoplasts generated from human polymorphonuclear leukocytes (PMNs) are vesicles consisting solely of cytoplasm surrounded by plasma membrane and devoid of granule activity remains to be tested. PMN cytoplasts were prepared by centrifugation of intact cells on a Ficoll step gradient in the presence of cytochalasin B. Two granule membrane markers, Mol, a fluorometrically detectable antigen, and cytochrome b, both of which have been shown to translocate to the plasma membrane during granule release, were compared for their activity in cytoplasts and intact PMNs. We found that the amount of Mol detected on the plasma membrane of intact PMNs, as compared with other membrane markers (such as antigens LFA-1 and beta 2m), increased 1.6-fold upon exposure of PMNs to Ficoll plus cytochalasin B prior to centrifugation. Another twofold increase in Mol expression occurred upon cytoplast preparation. Release of the granule enzymes, vitamin B12-binding protein, and lysozyme were also followed and correlated well (r = .78 and .92) with the amount of Mol antigen present on the cell surface. Cytochrome b was also found to be higher (1.4-fold) on plasma membranes isolated from cytoplasts than on plasma membranes isolated from intact control cells. These results indicate that some fusion of granule membranes and plasma membranes occurred during treatment of PMNs with Ficoll plus cytochalasin b and during cytoplast preparation.

The adhesion of neutrophils to vascular endothelium is an early event in their recruitment into acute inflammatory lesions. In evaluating potential neutrophil-endothelial adhesive mechanisms in acute inflammation, important considerations are that adhesion in vivo may occur very rapidly following injury and that the specificity of the reaction resides in altered endothelium. That is, neutrophils adhere only to altered endothelium adjacent to an inflammatory focus, rather than at random as would be expected if activation of neutrophils were the initiator of adhesion. We have explored a possible bridging role for complement in causing early neutrophil-endothelial cell adhesion. The complement system is involved in inflammatory processes, is capable of rapid amplification, and endothelial complement fixation at sites of inflammation could generate an endothelium-restricted signal for neutrophil adhesion. We have now developed a model in which this can be investigated without complicating factors such as immunoglobulin deposition, by constructing a novel molecule, a hybrid of the endothelial binding lectin Ulex europaeus I and of the complement activator cobra venom factor. This molecule has the capacity to cause fixation of complement on human umbilical vein endothelial cells. We show that complement fixation is a potent and rapid stimulus for neutrophil adhesion. Neutrophil adhesion requires only endothelial deposition of C3, and is mediated through the type 3 complement receptor.

Mo3e is a protease-sensitive Ag (p75,50) selectively expressed by human monocytic cells stimulated in vitro by exposure to various activating factors including PMA. Here, we report the existence of a large intracellular pool of Mo3e Ag in addition to that expressed on the surface of activated U-937 cells. As detected by quantitative immunofluorescence analysis, permeabilization of unstimulated and PMA-stimulated U-937 cells revealed a latent pool of Mo3e Ag that was 75-fold and 9-fold greater, respectively, than the magnitude of Mo3e Ag expressed on the surface. PMA stimulation not only induced an increase in the relative proportion of Mo3e antigen expressed on the surface membrane, but also stimulated a 1.8-fold increase in "total" Mo3e detectable in permeabilized cells. Trypsin treatment of intact PMA-stimulated U-937 cells eliminated surface Mo3e expression but had little measureable effect on the total Mo3e pool. Permeabilization also uncovered a sequestered compartment of Mo3e Ag in I-937 cells, a variant of U-937 that is surface Mo3e negative. Although the PMA-induced surface Mo3e expression of U-937 was abrogated by cycloheximide, the total pool of Mo3e detectable in permeabilized PMA-stimulated cells was only partially reduced; cycloheximide treatment caused no reduction in the intracellular Mo3e compartment of unstimulated U-937 cells. Detergent lysates of PMA-stimulated U-937 cells exhibited undiminished quantities (relative to untrypsinized cells) of p75 and p50 proteins immunoreactive with anti-Mo3e mAb as detected by Western blotting. This trypsin-sequestered intracellular Mo3e Ag may serve as a reservoir for the up-regulated surface expression of Mo3e that occurs as a result of mononuclear phagocyte activation.

A murine monoclonal IgM antibody recognizes an antigen (Mo3e) found on the surface of human peripheral blood monocytes stimulated with phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide and muramyl dipeptide and blocks the monocyte response to migration inhibitory factor (MIF). We utilized Western blot and immunoprecipitation analyses of whole cell lysates to investigate the biochemical nature of this monocyte antigen. Two distinct bands (75 kDa and 50 kDa) were detected by anti-Mo3e after monocyte lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transblotted onto nitrocellulose paper. Stimulation of monocytes with PMA resulted in a marked increase in the amount of the 50-kDa species. Immunoprecipitation of Mo3e from lysates of surface-iodinated cells demonstrated one broad band at 55-80 kDa which increased after PMA stimulation. The epitope identified by anti-Mo3e was resistant to 2-mercaptoethanol and heat treatment (100 degrees C/5 min) and was sensitive to trypsin or papain treatment. Two-dimensional SDS-PAGE analysis revealed that the 75-kDa species has an isoelectric point of 7.0 and the 50-kDa species is more acidic with an isoelectric point of 5.3. These results indicate that anti-Mo3e antibody defines unique monocyte proteins that may play a role as suggested by previous studies, in monocyte activation and responsiveness to MIF.

Mo1, LFA-1, and p150,95 are structurally related glycoproteins of the CD11/CD18 complex that are expressed on the membrane of human leukocytes. In the neutrophil, the surface expression of the CD11/CD18 complex is up-modulated (Mo1 greater than p150,95 much greater than LFA-1) by stimulatory factors that include calcium ionophore A23187, phorbol myristate acetate (PMA), and N-L-formyl-L-leucyl-L-phenylalanine (fMLP). Here, in an immunofluorescence analysis, we have examined CD11/CD18 glycoprotein expression by human monocytes, pulmonary alveolar macrophages (PAM, obtained by bronchoalveolar lavage), and breast milk macrophages (BMM) as compared to neutrophils before and after exposure to A23187 (1 microM), fMLP (0.1 microM), or PMA (0.1 microgram/ml) for 15 min at 37 degrees C. Unstimulated monocytes within unfractionated blood mononuclear cells kept at 4 degrees C (n = 13) expressed all three CD11/CD18 glycoproteins, and exposure to A23187 resulted in significant increases in the surface expression of Mo1 (median of 5.7-fold), LFA-1 (median of 2.1-fold), and p150,95 (median of 7.2-fold). Exposure to fMLP- or PMA-stimulated increases of lesser magnitude. CD11/CD18 expression by PAM (n = 9) was barely detectable and was unaffected by exposure to A23187. In contrast, BMM (n = 11) expressed all three CD11/CD18 glycoproteins (with considerable variability among specimens), but no increase was stimulated by A23187. These results demonstrate that monocytes, like neutrophils, have the capacity to respond to activating factors with an increase in CD11/CD18 glycoprotein expression; macrophage differentiation is accompanied by a loss (PAM) or retention (BMM) of CD11/CD18 expression that is unmodulated in response to activation.

Video-intensification fluorescence microscopy has been used to study the cell surface distribution of the complement receptor (CR) for C3bi (CR3) on human neutrophils. Fluorescein- or rhodamine-labeled monoclonal IgG or Fab fragments of antireceptor antibody were used as probes of receptor localization. C3bi receptors are uniformly distributed on untreated cells. Glass coverslips were coated with lipopolysaccharide (LPS) and serum was added; the serum deposits complement components, including C3bi, on the surface. When neutrophils were adherent to these coverslips, receptors were found in large clusters, and a fraction of the fluorescence remained uniform. Double-labeling studies were conducted by first labeling with anti-CR3 followed by attachment to LPS/serum-treated slides. This, in turn, was followed by labeling with the antibody conjugated to a second fluorophore. These studies revealed that the CR3 clusters were predominantly new antigenic sites exposed after attachment to the LPS/serum-treated slides. To determine the contribution of granule-associated CR3, we have studied neutrophils defective in receptor up-regulation, neutrophil cytoplasts, and a stimulator of granule release, A23187. Neutrophils from a patient with specific granule deficiency were found to be defective in granular CR3 and did not form clusters on C3-modified surfaces. The patient's neutrophils were defective in CR3 up-regulation and enzyme release as shown by fluorescence flow cytometry and gelatinase release, respectively. Cytoplasts also failed to show CR3 clusters on LPS/serum-treated coverslips. Furthermore, neutrophils treated with A23187 demonstrated numerous CR3 clusters. We suggest that formation of CR3 membrane domains during immune recognition requires the participation of intracellular granules. We speculate that these domains are formed by fusion of CR3-bearing granules at local sites of adhesion.

To identify the mechanisms accounting for hemodialysis-induced granulocytopenia, we undertook quantitative kinetic studies of a granulocyte-adhesion-promoting surface glycoprotein (Mo1). In eight patients undergoing maintenance hemodialysis, there was a fivefold increase in the mean cell-surface expression of Mo1 within 15 minutes after the start of dialysis with a new cuprophane membrane. The peak increase in surface Mo1 coincided with the maximal drop in neutrophil count and with the peak rise in the plasma levels of the complement-activation products C5a desArg and C3a desArg. During dialysis on a membrane being reused for the fifth time, no significant complement activation, no increase in Mo1 expression, and no change in neutrophil count were seen. C5a desArg (but not C3a desArg) induced a comparable increase in Mo1 expression on normal granulocytes in vitro at concentrations similar to those measured in vivo. Chemotactic peptide-induced granulocyte aggregation (a reflection of increased cell-to-cell adhesiveness) was specifically blocked by mouse monoclonal antibodies to Mo1 in vitro. These data suggest that the increased expression of Mo1 on granulocytes in vivo is in part mediated by C5a (and C5a desArg). The quantitative increase in granulocyte-surface Mo1 may provide a mechanism for initiating leukoaggregation, sequestration of granulocytes, and neutropenia during hemodialysis.

We hypothesized that neutrophil adhesion and lung injury could occur independent of the surface receptor glycoprotein, Mo1 (C3bi receptor). We investigated whether preincubation of human neutrophil-derived cytoplasts (cell fragments that lack nuclei and granules and have a fixed number of surface Mo1 receptors) with plasma and lipopolysaccharide (LPS) would augment the cytoplasts' ability to cause lung injury when activated. We also investigated whether preincubating normal human neutrophils treated with anti-Mo1 antibody with plasma and LPS would increase the neutrophils' ability to adhere and cause lung injury. Human neutrophils infused into isolated salt-perfused rat lungs subsequently stimulated with phorbol 12-myristate 13-acetate (PMA) resulted in lung injury as assessed by the accumulation of 125I-bovine serum albumin in the lung parenchyma. The infusion of cytoplasts resulted in significantly less injury. Cytoplasts preincubated in 20% human plasma and LPS caused an increase in lung injury. Similarly, neutrophils treated with plasma, LPS, and anti-Mo1 antibody or neutrophils congenitally deficient in the Mo1 surface receptor and treated with plasma and LPS augmented lung injury. Plasma and LPS preincubation also increased anti-Mo1 antibody-treated neutrophil adhesion to endothelial cell monolayers after activation by PMA. Thus, plasma and LPS increase adhesion and lung injury caused by neutrophils or neutrophil fragments that share defects in Mo1 receptor expression.

A dog with severe recurrent bacterial infections, impaired pus formation, delayed wound healing, and severe persistent leukocytosis was the result of a mother-son mating. Assessment of leukocyte function revealed profound abnormalities in adherence-dependent activities including impaired granulocyte adhesion to glass/plastic surfaces or nylon wool, decreased granulocyte aggregation and chemotaxis, and diminished lymphocyte blastogenesis, but normal neutrophil oxidative activity, serum immunoglobulin, and complement levels. By immunofluorescence analysis, CD11b and CD18 monoclonal antibodies specific for the 155-kd alpha polypeptide of Mo1 (gp 155, 94) and the 94 kd beta peptide common to Mo1, LFA-1 (gp 170, 94), and Leu M5 (p 150, 94) (surface molecules that promote leukocyte adhesion) failed to bind to unstimulated and A23187 calcium ionophore-stimulated granulocytes or mononuclear cells of the affected dog as compared with strong specific binding to canine control cells. The Mo1 glycoproteins were only barely detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of immunoprecipitates from lysates of 125I surface-labeled neutrophils from the affected dog as compared with intense bands seen with canine control cell precipitates. We conclude that this dog has a severe leukocyte surface glycoprotein deficiency syndrome that is similar, if not identical, to that recently recognized in humans. Dogs with deficiency of leukocyte Mo1, LFA-1, and Leu M5 expression may represent a useful animal model to characterize further the molecular basis for an inherited disorder in leukocyte effector function.

HL-60 promyelocytic cells acquire the surface expression of the Mo3e antigenic determinant after exposure to PMA or compounds that raise intracellular concentrations of cyclic AMP (dibutyryl cyclic AMP or a combination of cholera toxin and IBMX). The expression of Mo3e by these stimulated HL-60 cells coincides with the development of features of monocyte-macrophage differentiation (characteristic morphology, nonspecific esterase activity, and respiratory burst activity). During in vitro monocyte-macrophage differentiation, HL-60 cells become responsive to migration inhibitory factor (MIF); the MIF responsiveness of differentiated HL-60 cells is blocked by anti-Mo3e monoclonal antibody. These findings further support the relationship between the expression of Mo3e and the cellular response to MIF.

Fluorescence microscopy has been used to study the cell surface distribution of the complement receptor for C3bi (CR3) on human neutrophils during locomotion. CR3 is an integral membrane protein that participates in cell attachment phenomena including chemotaxis. Fluorescein- and rhodamine-conjugated monoclonal IgG or Fab fragments were used to label CR3. We have previously shown that CR3 is uniformly distributed on unstimulated cells. During cell locomotion the fluorescent labels redistribute to the uropod and retraction fibers. To better understand the role of CR3 in chemotaxis, we have performed sequential two-color labeling experiments in conjunction with fluorescence microscopy. Double-labeling experiments were conducted by labeling adherent neutrophils with fluorescein-conjugated anti-CR3 followed by chemotaxis in a gradient of FMLP (10(-7) M). The cells were then labeled again with rhodamine-conjugated anti-CR3. The uropod and distal training filopodia were labeled with fluorescein, whereas the cell body and occasionally proximal filopodia near the uropod were labeled with rhodamine. When neutrophils were fixed and permeabilized prior to the second CR3 labeling, the second fluorescent label was localized to a granule-like compartment(s), often near the lamellipodium. The results suggest a flow of CR3 from intracellular granules----lamellipodia and cell body----uropod----trailing filopodia during chemotaxis.

Monoclonal antibodies reactive with myeloid cell surface antigens were used to study evolutionary changes in granulocyte surface antigens from primate species. Certain of these granulocyte membrane antigens are conserved in phylogenetically distant species, indicating the potential functional importance of these structures. The degree of conservation of these antigens reflects the phylogenetic relationship between primate species. Furthermore, species of the same genus show similar patterns of binding to this panel of anti-human myeloid antibodies. This finding of conserved granulocyte surface antigens suggests that non-human primates may provide a model system for exploring uses of monoclonal antibodies in the treatment of human myeloid disorders.

Structural and functional features of a novel disorder characterized by recurrent bacterial infections are reviewed. This disease is associated with a number of phagocyte adhesion defects. In 10 patients, phenotypic analysis with monoclonal antibodies (MAb) revealed the same basic defect in all patients: deficiency of at least two leukocyte surface glycoproteins, Mo1 and LFA-1. These two antigens have distinct alpha subunits (Mo1 alpha = 155 kilodaltons, LFA-1 alpha = 177 kilodaltons) noncovalently linked to a common beta subunit (94 kilodaltons). Mo1 is closely associated with or identical to a receptor for the iC3b fragment of the third component of complement. LFA-1 is involved in lymphocyte proliferation, cytotoxicity, and natural killing. MAb directed to this family of glycoproteins induce functional defects in normal cells similar to those observed in deficient cells. In normal cells, the surface expression of these glycoproteins is regulated by the state of cell activation. Mitogens and alloantigens significantly increase the surface expression of LFA-1 on T lymphocytes. Stimuli that induce degranulation in neutrophils increase the surface expression of Mo1. In all patients with combined Mo1, LFA-1 deficiency, the predominant clinical manifestations were more characteristic of a phagocyte than a lymphocyte disorder. In vitro studies, however, reveal significant defects in phytohemagglutinin-induced proliferation that are more apparent at lower concentrations of the lectin. In some families, more than one sibling is affected. Intermediate levels of Mo1 were observed on granulocytes from both parents of one child. In one family, however, only the mother had significantly reduced levels of Mo1, which indicates heterogeneity in the inheritance of this disorder.

Mo1 is a glycoprotein heterodimer found on the surface of phagocytic cells. By use of monoclonal antibodies directed against various epitopes of the 155 kd alpha-chain of Mo1, two distinct functions of this glycoprotein have been identified. Mo1 serves as the receptor (CR3) for iC3b, one of the breakdown products of the third component of complement, and in addition appears to be involved in promoting cell-cell adhesion of granulocytes. In studies performed with a subline of the acute myelogenous leukemia tumor cell line KG1, we found cells from this subline (KG1m) incapable of iC3b-mediated binding despite these cells having surface Mo1. Five distinct epitopes on Mo1 identifiable on normal cells by a panel of anti-Mo1 alpha-chain monoclonal antibodies (including OKM10, thought to be identical to or close to the iC3b binding site) were equally present on KG1m by indirect immunofluorescence. The electrophoretic mobilities of both the alpha- and beta-chains of Mo1 derived from KG1m were identical to those isolated from normal granulocytes. To determine whether altered receptor mobility or decreased surface density of Mo1 was responsible for the lack of Mo1-related functions, binding to EiC3b of isolated Mo1 derived from KG1m lysate was measured. KG1m-derived Mo1 did not bind to EiC3b when compared with normal granulocyte-derived Mo1, suggesting that the lack of iC3b binding is not secondary to decreased surface receptor number or mobility. This subline of KG1 provides an excellent model for the study of the relationship between surface receptor structure and function.

Inasmuch as adenine nucleotides may be secreted by platelets during inflammation, we sought to determine whether ATP and related compounds could serve as stimuli of neutrophil (polymorphonuclear cells, PMN) activation as manifested by an increase in their adhesive properties. Exposure of isolated human PMN to ATP or its nonhydrolyzable analog, adenosine 5'-O-(3-thiotriphosphate) did indeed stimulate an increase in cellular adhesive function as assessed by an increase in the surface expression of the leukocyte adhesion-promoting glycoprotein, Mo1 (CD11b/CD18), the initiation of PMN aggregation, and (in the case of ATP) the attachment of increased numbers of albumin-coated polystyrene latex beads. However, this increase in PMN adhesive function was not accompanied by the generation of products of the respiratory burst. These in vitro data suggest the possible influence of secreted adenine nucleotides in promoting neutrophil adhesion-dependent interactions at inflammatory sites in vivo.

In our study of the human monocyte response to migration inhibitory factor (MIF), we screened murine anti-monocyte monoclonal antibodies (MAb) for their ability to affect the MIF response. When blood monocytes were preincubated for 30 min at 37 degrees C with MAb of various cell specificities, it was found that Mo3e-MAb could inhibit the monocyte response to MIF. This effect was observed at an antibody concentration of 0.37 microgram/10(6) monocytes. This blocking activity appeared to be specific for Mo3e-MAb, since MAb specific for Mo1, Mo2, Mo4, alveolar macrophage (PAM-1), and platelet (Plt-1) antigens were unable to block the MIF response. Specificity was also shown by experiments performed with MAb similar to Mo3e-MAb in their immunofluorescence staining patterns. Two MAb of this type were not able to block the MIF response. All of these MAb are of the IgM isotype, and therefore it is unlikely that the blocking activity of Mo3e-MAb is mediated by the Fc receptor. These studies suggest that Mo3e-MAb may represent an anti-MIF receptor antibody. Mo3e-MAb recognizes a protease-sensitive antigen of 80 kD and 50 kD that is weakly expressed on fresh blood monocytes but is up-modulated by stimulation of monocytes with bacterial lipopolysaccharide, muramyl dipeptide, or phorbol compounds. Mo3e is also expressed by human myeloid cell lines, U-937 and HL-60, that have been stimulated in culture by biologically active phorbol diester compounds.

A case of a 30-year-old black woman who developed acute, extensive intraoral hyperpigmentation is reported. Its relationship to previously described entities is discussed. The condition was associated with the presence of melanophages both in the submucosa and in the oral epithelium. The melanophages displayed phenotypic markers of a population of intraepidermal melanophages previously described as potent T cell-activating, antigen presenting cells (T6- DR+ OKM5+ OKM1-) and of activated monocytes (Mo3e+), as well as other monocyte-macrophage markers (Mono 1 and My7). The distinct phenotype of these melanophages suggests that they could play an active role in the promotion of inflammatory disease.

Mo1 and gp160-gp93 are two surface membrane glycoprotein heterodimers present on granulocytes and monocytes derived from humans and guinea pigs, respectively. We purified both antigens and found that their alpha subunits had identical N-termini which were significantly homologous to the alpha subunit of the human adhesion platelet glycoprotein IIb/IIIa.

Deficiency of a granulocyte surface glycoprotein of 150,000-D had been associated with defective C3- and IgG-dependent phagocytosis in a patient with recurrent bacterial infections. By using monoclonal antibodies, we found that this patient's granulocytes, monocytes, and null cells were deficient in Mo1 (equivalent to OKM1 and Mac-1), a cell surface molecule consisting of two noncovalently linked glycoproteins of 155,000 and 94,000 D. The 155,000-D subunit is closely associated with the human complement receptor that recognizes C3bi and/or a further degradation product termed C3dg (C3bi receptor); the 94,000-D subunit has been shown to be shared, on normal cells, by two other surface membrane glycoproteins: lymphocyte function-associated antigen-1 (LFA-1) and P-150, 95. Both subunits of Mo1 were deficient on the patient's granulocytes as determined by immunoprecipitation with subunit-specific monoclonal antibodies as well as fluorescence analysis. Mol-deficient monocytes, like granulocytes, had defective C3-and IgG-dependent phagocytosis. Natural killing activity by the patient's peripheral blood leukocytes was normal. Mo1-deficient granulocytes and monocytes rosetted normally with sheep erythrocytes coated with C3bi. This rosetting was totally inhibited by a mixture of anti-Mo1 and anti-C3b (the major fragment of C3) receptor antibodies but not by either antibody alone. Since monoclonal antibodies to the 155,000-D subunit of Mo1 can inhibit C3bi receptor binding, immune phagocytosis, opsonized zymosan-induced degranulation, and superoxide generation by normal phagocytes (functions which are defective in Mo1-deficient cells), it appears likely that Mo1 deficiency may in part underlie the functional aberrations leading to recurrent bacterial infections in man.