Relationship Between Monocytosis and T-Lymphocyte Function in Human Cancer 1,2 Gary W. Wood,
3
James E. Neff,4 and Ronald Stephens 4
ABSTRACT-This study was designed to: 1) determine the relative number of monocytes In mononuclear cell suspensions derived from the peripheral blood of cancer patients and 2) ascertain If any relationship existed between the numbers of monocytes In those cell suspensions and T-lymphocyte function. Monocytes were quantitated by morphology verified by phagocytosis of antibody-coated erythrocytes. A significant difference (~O.01) existed between the number of monocytes In suspensions from normal Individuals (26.2±4.3) and cancer patients (38.0±13.4). The cancer petlents were divided Into 2 groups: 1) those who exhibited normal In vitro T-cell responses to phytohemagglutinin and 2) those In whom responses were significantly suppressed. The mean number of monocytes In suspensions from the cancer patient group with normal responses was 29±9, whereas that from the cancer patients with suppressed responses was 47±11, a highly significant difference (P:50.01). Therefore, the study demonstrated two things: 1) Mononuclear cell suspensions derived from cancer patients exhibited significant monocytosis relative to those from normal Individuals and 2) a strong correlation existed between monocytosis and suppressed T-lymphocyte function In vltro.-JNCI 83: 587-592, 1979.
Several studies in which different methodologies have been used have established that approximately one-fifth to one-third of the mononuclear cells in suspensions derived from peripheral blood of normal individuals by Ficoll-Hypaque density gradient sedimentation are characterized as mononuclear phagocytes (1-3). Considerable evidence now shows that cells of monocyte-macrophage lineage perform an essential accessory role in the immune response. Present knowledge of the function of macrophages in antigen presentation, initiation of T-cell responses, cell cooperation, and immune suppression has been reviewed by several workers (4-7). Heightened interest in the functional significance of circulating mononuclear phagocytes has led us to study monocytes in mononuclear cell suspensions from the peripheral blood of cancer patients. T-Iymphocyte dysfunction is not an uncommon finding in patients with malignant tumors and is generally, but not necessarily, associated with advanced disease (8). Altered immunocompetence may be detected in vivo through the failure of patients to manifest specific delayed hypersensitivity skin test reactions to a battery of antigens or in vitro through reduced cellular responses to polyclonal T-Iymphocyte mitogens such as PHA and concanavalin A, reduced mixed leukocyte culture responses, or blocked cellmediated cytotoxicity. What was observed has often been assumed to be an acquired T-cell defect, but more recently, the functional capabilities of T-cells have been shown to be susceptible to modulation by various extrinsic factors, both cellular and humoral; i.e., the suppression may be observed either in the presence or
587 Downloaded from https://academic.oup.com/jnci/article-abstract/63/3/587/1056406 by University of Durham user on 03 March 2018
absence of autologous serum or factors derived from serum. Several recent studies have implicated cells of the mononuclear phagocyte system in suppression of cellmediated immunity (9-14). Some of those studies have implicated macrophages in .immunosuppression associated with tumor progression (9-13); recently, peripheral blood monocytes from cancer patients have been reported to be capable of suppressing the proliferative response to polyclonal T-cell mitogens (9, 13). Recently, the role of various suppressor cells in cancer was reviewed by Kirchner (15). It was apparent from those studies that modulation of T-cell function by mononuclear phagocytes was at least partially dependent on their number and/or activation level (14). Thus we designed the present study to enumerate monocytes in mononuclear cell suspensions derived from the peripheral blood of cancer patients by FicollHypaque density gradient sedimentation and then to determine if any correlation existed between the numbers of monocytes and T-cell function in the separated cell populations.
MATERIALS AND METHODS
Populations of patients. -Seventy-eight patients with nonlymphoreticular cancers were included. Most patients (61/78) had bone tumors of various histologic types. Because the present study was mainly explorative, the patients were not categorized further, i.e., as to extent of disease. Although some of the patients with tumors other than those of the bone were on various radiation and chemotherapeutic regimens, all bone tumor patients were studied prior to surgical removal of their tumors and prior to institution of therapy. Normal healthy adults were included as controls for
ABBREVIATIONS USED: cpm=counts per minute; EAC=erythrocyteantibody-complement; FITC = fluorescein isothiocyanate; HBSS = Hanks' balanced salt solution; PBS = phosphate-buffered saline; PHA=phytohemagglutinin; SRBC=sheep red blood cells; VCB= Veronal gelatin buffer.
1 Received August 29, 1978; revised March 7, 1979; accepted March 13, 1979. 2 Supported in part by Public Health Service grant CAI9333 from the National Cancer Institute. 3 Department of Pathology, College of Health Science and Hospital, University of Kansas Medical 'Center, 39th St. and Rainbow Blvd., Kansas City, Kan. 66103. 4 Department of Surgery, College of Health Science and Hospital, University of Kansas Medical Center.
JNCI, VOL. 63, NO.3, SEPTEMBER 1979
588 Wood, Neff, and Stephens the separation procedure, cell enumeration, and T-cell function studies. Cell separation.-Peripheral venous blood was drawn into Vacutainer tubes containing 250 V of preservativefree heparin. Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density gradient sedimentation (16). Cells removed from the plasma-Ficoll-Hypaque interphase were washed with HBSS. Routinely, the cell population thus obtained was evaluated without further modifications. T-lymphocyte quantitation.- The total T-lymphocyte population was enumerated by a standard erythrocyte rosette assay (17). SRBC (Colorado Serum Co., Denver, Colo.), stored in Alsever's solution at 4° C for no more than I week, were washed three times with VGB and resuspended to 0.5% in HBSS. The SRBC were mixed with an equal volume of mononuclear cells (I.OXI06 /ml), incubated at 37° C for 5 minutes, and sedimented at 300Xg for 5 minutes. The sedimented cells were incubated at 4° C for 18 hours and gently resuspended. We determined the percentage of cells forming rosettes consisting of at least three attached erythrocytes by counting at least 200 total cells. B-lymphocyte quantitation. -B-cells were quantitated by anti-IgM-IgD immunofluorescence. Rabbit antihuman IgM (Il-chain-specific; Meloy Laboratories Inc., Springfield, Va.) and rabbit antihuman IgD (6chain-specific; Hyland Division, Travenol Laboratories, Inc., Costa Mesa, Calif.) were purchased, and the globulin fractions were obtained by 1.8-M ammonium sulfate precipitation and conjugated with FITC as described previously (18). The fluoresceinated reagents were specific for IgM and IgD as determined by Ouchterlony gel diffusion analysis. The molar fluorescein: protein ratios of the reagents varied between 3.5 and 4.5. Antisera were disaggregated prior to use by centrifugation at 145,000Xg for 60 minutes. Direct immunofluorescence assays were performed as described previously (19). FITC anti-IgM (20 Ill) and FITC anti-IgD (20 Ill) were mixed with 5.0XlOs cells in 0.1 ml of Dulbecco's PBS (pH 7.2), incubated for 20 minutes at 4° C, washed two times with ice-cold PBS, and mounted with 90% glycerol-IO% PBS. The percentage of cells exhibiting the characteristic pattern of B-lymphocyte surface immunofluorescence was determined after at least 200 total cells had been counted. A Zeiss fluorescence microscope equipped with an HBO 200 mercury vapor light source, Schott 65 and 50 barrier filters, and Schott BGl2 exciter filters was used. Monocyte quantitation.-For simplicity and because it is a reliable method for. detecting and quantitating mononuclear phagocytes, we chose to measure these phagocytes strictly by morphology in cytocentrifuged, Wright's-stained preparations. We took a conservative approach in that only obvious monocytes were counted as such, e.g., cells with a high cytoplasm-to-nucleus ratio and convoluted nuclei. Furthermore, extensive control experiments were performed to establish that the percentage of monocytes, as determined by morphologic evaluation, was close to the percentage of JNCI, VOL. 63, NO.3, SEPTEMBER 1979 Downloaded from https://academic.oup.com/jnci/article-abstract/63/3/587/1056406 by University of Durham user on 03 March 2018
cells capable of phagocytosing sensitized indicator cells as described below. IgG EAC phagocytosis assay.-C3 and/or Fe receptor-bearing cells were detected by the IgG EAC rosette method (20). A 5% suspension of washed SRBC was mixed with equal volumes of rabbit IgG antiSRBC (diluted to contain I agglutinating V/ml) and appropriately diluted human complement. The mixture was incubated for 30 minutes at 37° C, washed three times with VGB, and reconstituted to 0.5% with HBSS. We performed the rosette assay by mixing equal volumes (0.25 ml) of sensitized erythrocytes and mononuclear cells (2X106 cells/ml), incubating the mixture for 5 minutes at 37° C, and sedimenting the mixture for 5 minutes at 300Xg. The assay was performed four times. Cells in two of the tubes were resuspended, and the percentage of rosettes was determined as described previously for erythrocyte rosettes. The remaining tubes were reincubated at 37° C for 60 minutes to facilitate erythrocytophagy, and the cells were resuspended with a Pasteur pipette, cytocentrifuged, and stained with Wright's agent. The method for analysis of the cytocentrifuged cells was critical. The purpose was to perform a combined morphologic-functional analysis of the rosetted tells. Rosette-positive cells were counted and placed into two categories: I) nonphagocytic cells with a high nucleusto-cytoplasm ratio and 2) phagocytic cells. More than SOO rosette-positive cells were counted for each determination. T-lymphocyte function assay.-A standard PHA stimulation assay was performed. Mononuclear cells were suspended in Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 100 V penicillin/ml, and 100 Ilg streptomycin/ml. Cells (5XI0 4 ) in 0.2 ml of medium were added to each well of 96-well, flat-bottomed Linbro Dispose-trays (IS-FB-96TC; Linbro Scientific Inc., Hamden, Conn.). PHA-P (purified) (GIBCO, Grand Island, N.Y.) at five different concentrations (I, 2, 5, 10, and 25 liters of stock PHA-P/ml) was added to the wells in 10 liters of HBSS. Six replicates were performed for each mitogen concentration as well as for the cell control. Cells were cultured for 72 hours in 5% CO2 in a humidified incubator at 37° C, 12.6 Ci of eH]thymidine was added to each well, and the culture was continued for 18 hours. Samples were harvested on filter paper with a MASH II automated cell harvester (Microbiological Associates, Bethesda, Md.), solubilized, and prepared for counting with Aquasol (New England Nuclear, Boston, Mass.). All samples were counted in a Mark III beta scintillation counter (Searle Analytic, Des Plaines, Ill. ). Statistics. - The significance of the differences between groups of data was determined by means of the Wilcoxon rank sum test.
RESULTS Normal Controls The initial aim of the study was to enumerate mono-
Monocytosis and Immunosuppression In Cancer 589 cytes in mononuclear cell suspensions derived from cancer patients. To do that, first we had to establish normal quantitative values for this laboratory for Tlymphocytes, B-lymphocytes, and monocytes in mononuclear cell suspensions derived by Ficoll-Hypaque sedimentation, because regional or technical differences could exist between laboratories. The average number of T-cells detected in one representative series of 31 determinations was 62.2±6.4%, with a range of 51-73%. The mean value for B-lymphocytes in one series of 46 determinations on normal individuals was 9.4±4.0, with a range of 3-20%. The B-cells were enumerated by IgM-IgD immunofluorescence. Monocytes may be quantitated by latex phagocytosis, nonspecific esterase staining, or morphology, but each of these methods must be performed carefully. In the present study we used morphology as the sole defining criterion for monocytes; to verify that morphologic assessment was reliable, we performed the following experiment: The mononuclear cells from normal human peripheral blood were rosetted with IgG EAC, incubated for 60 minutes to allow erythrocytophagy, cytocentrifuged, and stained. Under those conditions all monocvtes formed rosettes, as did a proportion of the lymphocytes. Most monocytes phagocytized the opsonized erythrocytes. The identity of the rosettepositive lymphocytes, e.g., T-cell, L-cells, and B-cells, was essentially irrelevant; these cells were simply counted as nonmonocytes. We determined the proportion of the rosetted cells comprised by monocytes by counting phagocytic cells and morphologically obvious nonphagocytic monocytes. That fraction then was multiplied by the fraction of the total population that was IgG EAC rosette-positive, which thus gave the percentage of the total population comprised by monocytes. That value then was compared with the percentage of monocytes that had been determined by morphologic analysis alone. The results of those experiments are given in table l. Clearly, from those data TABLE l.-Quantitation of mononuclear phagocytes in human peripheral blood by IgG EAC phagocytosis and morphology" IgG EAC rosette-positive monocytes Measurement Mean±SE Variance Range
Phagocytic"
Nonphagocytic"
Total
Monocytes by morphology"
23.3±5.6 7.5 8-40
5.2±1.2 3.4 2-17
28.1±3.2 7.1 16-44
26.2±4.3 6.6 18-38
a Data were obtained from analyses on peripheral blood mononuclear cells from 31 apparently normal, human volunteers. b The percentage of the total cell population comprised by phagocytic monocytes was obtained by determination of the percent of IgG EAC rosette-positive cells that phagocytized erythrocytes and multiplication of that number by the percent of total cells rosetted. A portion of the rosetted cells was morphologically monocytic but nonphagocytic within the 60-min incubation period. The percentage was determined as described in footnote b for phagocytic cells. d Obvious monocytes (low nucleus-to-cytoplasm ratio and convoluted nuclei) were counted following cytocentrifugation and staining with Wright's agent. C
the number of cells determined by the two methods was essentially the same, which thus verifies that monocytes could be quantitated with the use of morphology.
Cell Quantltatlon in Cancer Patients Moderate decreases in the numbers of circulating Tlymphocytes in cancer patients have been well documented. We observed somewhat depressed T-cell numbers in the mononuclear cell suspensions of many patients, and the major concern was the identification of the erythrocyte rosette-negative cell population, e.g., the contribution of two readily identifiable erythrocyterosette-negative cell types: B-lymphocytes and monocytes. Mononuclear cell suspensions from cancer patients were compared to those from normal individuals; both cell suspensions were tested over a similar timespan. Two major observations were made with respect to those data: I) In general, the contribution of B-lymphocytes to the non-T-cell pool was lower in cancer patients (mean, 5.3±4.6; range, 0-18) than that in normal individuals (mean, 9.4±4.0; range, 3-20). Many cancer patients had less than 1% B-cells in the cell suspensions. 2) The percentage of monocytes was significantly greater (P;::::O.OI) in cell populations from cancer patients (mean, 38.0±14.2; range, 10-66) than in those from normal individuals (mean, 26.2±4.3; range 18-38). Furthermore, in most patients greater than 95% of the cells could be accounted for as T-cells (rosette-positive), B-cells (IgM-IgD-positive), or monocytes (table 2). When significant numbers of cells could not be accounted for, determination of the nature of those cells was not possible. A few patients had significant numbers of granulocytes in the separated cell populations, but generally granulocytes represented less than 3% of the total cells. Clearly, in all mononuclear cell suspensions tested, monocytes comprised the major proportion of the mononuclear non-T-cells.
Relationship Between Monocyte Levels and T-Lymphocyte Function Polyclonal T-Iymphocyte function was assessed in mononuclear cell populations by the measurement of the mitogenic response to PHA. All responses were measured after 72 hours of culture in the presence of PHA. Five concentrations of PHA ranging from undiluted stock PHA-P to a 1:25 dilution were used in all tests. Determination of an accurate mechanism for expression of the results was a major problem because of the intrinsic variability of the mitogen stimulation method. Maximum mean stimulation values always were selected, because stimulation profiles vary from individual to individual; e.g., one individual's cells may stimulate maximally at the highest mitogen concentration, whereas another's may do so at the lowest. In this series of experiments, the responses of cells from a normal individual and a cancer patient always were tested on the same day and compared. Because of JNCI, VOL. 63, NO.3,
Downloaded from https://academic.oup.com/jnci/article-abstract/63/3/587/1056406 by University of Durham user on 03 March 2018
SEPTEMBER 1979
590 Wood, Neff, and Stephens TABLE 2.-Contribution of T-cells. Bscells, and monocytes to mono-
TABLE 3.-Representative mitogen stimulation data on mononu-
nuclear cell suspensions derived from an unselected series of cancer patients
clear cells from cancer patients
T-lymphocytes"
B-lymphocytes b
2
33 18 32 40
4
44
4
23 63 21 16 28 48 35 64 58 64 51 48 53 53 60
63 52 69 60 51 71
3
James E. Neff,4 and Ronald Stephens 4
ABSTRACT-This study was designed to: 1) determine the relative number of monocytes In mononuclear cell suspensions derived from the peripheral blood of cancer patients and 2) ascertain If any relationship existed between the numbers of monocytes In those cell suspensions and T-lymphocyte function. Monocytes were quantitated by morphology verified by phagocytosis of antibody-coated erythrocytes. A significant difference (~O.01) existed between the number of monocytes In suspensions from normal Individuals (26.2±4.3) and cancer patients (38.0±13.4). The cancer petlents were divided Into 2 groups: 1) those who exhibited normal In vitro T-cell responses to phytohemagglutinin and 2) those In whom responses were significantly suppressed. The mean number of monocytes In suspensions from the cancer patient group with normal responses was 29±9, whereas that from the cancer patients with suppressed responses was 47±11, a highly significant difference (P:50.01). Therefore, the study demonstrated two things: 1) Mononuclear cell suspensions derived from cancer patients exhibited significant monocytosis relative to those from normal Individuals and 2) a strong correlation existed between monocytosis and suppressed T-lymphocyte function In vltro.-JNCI 83: 587-592, 1979.
Several studies in which different methodologies have been used have established that approximately one-fifth to one-third of the mononuclear cells in suspensions derived from peripheral blood of normal individuals by Ficoll-Hypaque density gradient sedimentation are characterized as mononuclear phagocytes (1-3). Considerable evidence now shows that cells of monocyte-macrophage lineage perform an essential accessory role in the immune response. Present knowledge of the function of macrophages in antigen presentation, initiation of T-cell responses, cell cooperation, and immune suppression has been reviewed by several workers (4-7). Heightened interest in the functional significance of circulating mononuclear phagocytes has led us to study monocytes in mononuclear cell suspensions from the peripheral blood of cancer patients. T-Iymphocyte dysfunction is not an uncommon finding in patients with malignant tumors and is generally, but not necessarily, associated with advanced disease (8). Altered immunocompetence may be detected in vivo through the failure of patients to manifest specific delayed hypersensitivity skin test reactions to a battery of antigens or in vitro through reduced cellular responses to polyclonal T-Iymphocyte mitogens such as PHA and concanavalin A, reduced mixed leukocyte culture responses, or blocked cellmediated cytotoxicity. What was observed has often been assumed to be an acquired T-cell defect, but more recently, the functional capabilities of T-cells have been shown to be susceptible to modulation by various extrinsic factors, both cellular and humoral; i.e., the suppression may be observed either in the presence or
587 Downloaded from https://academic.oup.com/jnci/article-abstract/63/3/587/1056406 by University of Durham user on 03 March 2018
absence of autologous serum or factors derived from serum. Several recent studies have implicated cells of the mononuclear phagocyte system in suppression of cellmediated immunity (9-14). Some of those studies have implicated macrophages in .immunosuppression associated with tumor progression (9-13); recently, peripheral blood monocytes from cancer patients have been reported to be capable of suppressing the proliferative response to polyclonal T-cell mitogens (9, 13). Recently, the role of various suppressor cells in cancer was reviewed by Kirchner (15). It was apparent from those studies that modulation of T-cell function by mononuclear phagocytes was at least partially dependent on their number and/or activation level (14). Thus we designed the present study to enumerate monocytes in mononuclear cell suspensions derived from the peripheral blood of cancer patients by FicollHypaque density gradient sedimentation and then to determine if any correlation existed between the numbers of monocytes and T-cell function in the separated cell populations.
MATERIALS AND METHODS
Populations of patients. -Seventy-eight patients with nonlymphoreticular cancers were included. Most patients (61/78) had bone tumors of various histologic types. Because the present study was mainly explorative, the patients were not categorized further, i.e., as to extent of disease. Although some of the patients with tumors other than those of the bone were on various radiation and chemotherapeutic regimens, all bone tumor patients were studied prior to surgical removal of their tumors and prior to institution of therapy. Normal healthy adults were included as controls for
ABBREVIATIONS USED: cpm=counts per minute; EAC=erythrocyteantibody-complement; FITC = fluorescein isothiocyanate; HBSS = Hanks' balanced salt solution; PBS = phosphate-buffered saline; PHA=phytohemagglutinin; SRBC=sheep red blood cells; VCB= Veronal gelatin buffer.
1 Received August 29, 1978; revised March 7, 1979; accepted March 13, 1979. 2 Supported in part by Public Health Service grant CAI9333 from the National Cancer Institute. 3 Department of Pathology, College of Health Science and Hospital, University of Kansas Medical 'Center, 39th St. and Rainbow Blvd., Kansas City, Kan. 66103. 4 Department of Surgery, College of Health Science and Hospital, University of Kansas Medical Center.
JNCI, VOL. 63, NO.3, SEPTEMBER 1979
588 Wood, Neff, and Stephens the separation procedure, cell enumeration, and T-cell function studies. Cell separation.-Peripheral venous blood was drawn into Vacutainer tubes containing 250 V of preservativefree heparin. Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density gradient sedimentation (16). Cells removed from the plasma-Ficoll-Hypaque interphase were washed with HBSS. Routinely, the cell population thus obtained was evaluated without further modifications. T-lymphocyte quantitation.- The total T-lymphocyte population was enumerated by a standard erythrocyte rosette assay (17). SRBC (Colorado Serum Co., Denver, Colo.), stored in Alsever's solution at 4° C for no more than I week, were washed three times with VGB and resuspended to 0.5% in HBSS. The SRBC were mixed with an equal volume of mononuclear cells (I.OXI06 /ml), incubated at 37° C for 5 minutes, and sedimented at 300Xg for 5 minutes. The sedimented cells were incubated at 4° C for 18 hours and gently resuspended. We determined the percentage of cells forming rosettes consisting of at least three attached erythrocytes by counting at least 200 total cells. B-lymphocyte quantitation. -B-cells were quantitated by anti-IgM-IgD immunofluorescence. Rabbit antihuman IgM (Il-chain-specific; Meloy Laboratories Inc., Springfield, Va.) and rabbit antihuman IgD (6chain-specific; Hyland Division, Travenol Laboratories, Inc., Costa Mesa, Calif.) were purchased, and the globulin fractions were obtained by 1.8-M ammonium sulfate precipitation and conjugated with FITC as described previously (18). The fluoresceinated reagents were specific for IgM and IgD as determined by Ouchterlony gel diffusion analysis. The molar fluorescein: protein ratios of the reagents varied between 3.5 and 4.5. Antisera were disaggregated prior to use by centrifugation at 145,000Xg for 60 minutes. Direct immunofluorescence assays were performed as described previously (19). FITC anti-IgM (20 Ill) and FITC anti-IgD (20 Ill) were mixed with 5.0XlOs cells in 0.1 ml of Dulbecco's PBS (pH 7.2), incubated for 20 minutes at 4° C, washed two times with ice-cold PBS, and mounted with 90% glycerol-IO% PBS. The percentage of cells exhibiting the characteristic pattern of B-lymphocyte surface immunofluorescence was determined after at least 200 total cells had been counted. A Zeiss fluorescence microscope equipped with an HBO 200 mercury vapor light source, Schott 65 and 50 barrier filters, and Schott BGl2 exciter filters was used. Monocyte quantitation.-For simplicity and because it is a reliable method for. detecting and quantitating mononuclear phagocytes, we chose to measure these phagocytes strictly by morphology in cytocentrifuged, Wright's-stained preparations. We took a conservative approach in that only obvious monocytes were counted as such, e.g., cells with a high cytoplasm-to-nucleus ratio and convoluted nuclei. Furthermore, extensive control experiments were performed to establish that the percentage of monocytes, as determined by morphologic evaluation, was close to the percentage of JNCI, VOL. 63, NO.3, SEPTEMBER 1979 Downloaded from https://academic.oup.com/jnci/article-abstract/63/3/587/1056406 by University of Durham user on 03 March 2018
cells capable of phagocytosing sensitized indicator cells as described below. IgG EAC phagocytosis assay.-C3 and/or Fe receptor-bearing cells were detected by the IgG EAC rosette method (20). A 5% suspension of washed SRBC was mixed with equal volumes of rabbit IgG antiSRBC (diluted to contain I agglutinating V/ml) and appropriately diluted human complement. The mixture was incubated for 30 minutes at 37° C, washed three times with VGB, and reconstituted to 0.5% with HBSS. We performed the rosette assay by mixing equal volumes (0.25 ml) of sensitized erythrocytes and mononuclear cells (2X106 cells/ml), incubating the mixture for 5 minutes at 37° C, and sedimenting the mixture for 5 minutes at 300Xg. The assay was performed four times. Cells in two of the tubes were resuspended, and the percentage of rosettes was determined as described previously for erythrocyte rosettes. The remaining tubes were reincubated at 37° C for 60 minutes to facilitate erythrocytophagy, and the cells were resuspended with a Pasteur pipette, cytocentrifuged, and stained with Wright's agent. The method for analysis of the cytocentrifuged cells was critical. The purpose was to perform a combined morphologic-functional analysis of the rosetted tells. Rosette-positive cells were counted and placed into two categories: I) nonphagocytic cells with a high nucleusto-cytoplasm ratio and 2) phagocytic cells. More than SOO rosette-positive cells were counted for each determination. T-lymphocyte function assay.-A standard PHA stimulation assay was performed. Mononuclear cells were suspended in Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 100 V penicillin/ml, and 100 Ilg streptomycin/ml. Cells (5XI0 4 ) in 0.2 ml of medium were added to each well of 96-well, flat-bottomed Linbro Dispose-trays (IS-FB-96TC; Linbro Scientific Inc., Hamden, Conn.). PHA-P (purified) (GIBCO, Grand Island, N.Y.) at five different concentrations (I, 2, 5, 10, and 25 liters of stock PHA-P/ml) was added to the wells in 10 liters of HBSS. Six replicates were performed for each mitogen concentration as well as for the cell control. Cells were cultured for 72 hours in 5% CO2 in a humidified incubator at 37° C, 12.6 Ci of eH]thymidine was added to each well, and the culture was continued for 18 hours. Samples were harvested on filter paper with a MASH II automated cell harvester (Microbiological Associates, Bethesda, Md.), solubilized, and prepared for counting with Aquasol (New England Nuclear, Boston, Mass.). All samples were counted in a Mark III beta scintillation counter (Searle Analytic, Des Plaines, Ill. ). Statistics. - The significance of the differences between groups of data was determined by means of the Wilcoxon rank sum test.
RESULTS Normal Controls The initial aim of the study was to enumerate mono-
Monocytosis and Immunosuppression In Cancer 589 cytes in mononuclear cell suspensions derived from cancer patients. To do that, first we had to establish normal quantitative values for this laboratory for Tlymphocytes, B-lymphocytes, and monocytes in mononuclear cell suspensions derived by Ficoll-Hypaque sedimentation, because regional or technical differences could exist between laboratories. The average number of T-cells detected in one representative series of 31 determinations was 62.2±6.4%, with a range of 51-73%. The mean value for B-lymphocytes in one series of 46 determinations on normal individuals was 9.4±4.0, with a range of 3-20%. The B-cells were enumerated by IgM-IgD immunofluorescence. Monocytes may be quantitated by latex phagocytosis, nonspecific esterase staining, or morphology, but each of these methods must be performed carefully. In the present study we used morphology as the sole defining criterion for monocytes; to verify that morphologic assessment was reliable, we performed the following experiment: The mononuclear cells from normal human peripheral blood were rosetted with IgG EAC, incubated for 60 minutes to allow erythrocytophagy, cytocentrifuged, and stained. Under those conditions all monocvtes formed rosettes, as did a proportion of the lymphocytes. Most monocytes phagocytized the opsonized erythrocytes. The identity of the rosettepositive lymphocytes, e.g., T-cell, L-cells, and B-cells, was essentially irrelevant; these cells were simply counted as nonmonocytes. We determined the proportion of the rosetted cells comprised by monocytes by counting phagocytic cells and morphologically obvious nonphagocytic monocytes. That fraction then was multiplied by the fraction of the total population that was IgG EAC rosette-positive, which thus gave the percentage of the total population comprised by monocytes. That value then was compared with the percentage of monocytes that had been determined by morphologic analysis alone. The results of those experiments are given in table l. Clearly, from those data TABLE l.-Quantitation of mononuclear phagocytes in human peripheral blood by IgG EAC phagocytosis and morphology" IgG EAC rosette-positive monocytes Measurement Mean±SE Variance Range
Phagocytic"
Nonphagocytic"
Total
Monocytes by morphology"
23.3±5.6 7.5 8-40
5.2±1.2 3.4 2-17
28.1±3.2 7.1 16-44
26.2±4.3 6.6 18-38
a Data were obtained from analyses on peripheral blood mononuclear cells from 31 apparently normal, human volunteers. b The percentage of the total cell population comprised by phagocytic monocytes was obtained by determination of the percent of IgG EAC rosette-positive cells that phagocytized erythrocytes and multiplication of that number by the percent of total cells rosetted. A portion of the rosetted cells was morphologically monocytic but nonphagocytic within the 60-min incubation period. The percentage was determined as described in footnote b for phagocytic cells. d Obvious monocytes (low nucleus-to-cytoplasm ratio and convoluted nuclei) were counted following cytocentrifugation and staining with Wright's agent. C
the number of cells determined by the two methods was essentially the same, which thus verifies that monocytes could be quantitated with the use of morphology.
Cell Quantltatlon in Cancer Patients Moderate decreases in the numbers of circulating Tlymphocytes in cancer patients have been well documented. We observed somewhat depressed T-cell numbers in the mononuclear cell suspensions of many patients, and the major concern was the identification of the erythrocyte rosette-negative cell population, e.g., the contribution of two readily identifiable erythrocyterosette-negative cell types: B-lymphocytes and monocytes. Mononuclear cell suspensions from cancer patients were compared to those from normal individuals; both cell suspensions were tested over a similar timespan. Two major observations were made with respect to those data: I) In general, the contribution of B-lymphocytes to the non-T-cell pool was lower in cancer patients (mean, 5.3±4.6; range, 0-18) than that in normal individuals (mean, 9.4±4.0; range, 3-20). Many cancer patients had less than 1% B-cells in the cell suspensions. 2) The percentage of monocytes was significantly greater (P;::::O.OI) in cell populations from cancer patients (mean, 38.0±14.2; range, 10-66) than in those from normal individuals (mean, 26.2±4.3; range 18-38). Furthermore, in most patients greater than 95% of the cells could be accounted for as T-cells (rosette-positive), B-cells (IgM-IgD-positive), or monocytes (table 2). When significant numbers of cells could not be accounted for, determination of the nature of those cells was not possible. A few patients had significant numbers of granulocytes in the separated cell populations, but generally granulocytes represented less than 3% of the total cells. Clearly, in all mononuclear cell suspensions tested, monocytes comprised the major proportion of the mononuclear non-T-cells.
Relationship Between Monocyte Levels and T-Lymphocyte Function Polyclonal T-Iymphocyte function was assessed in mononuclear cell populations by the measurement of the mitogenic response to PHA. All responses were measured after 72 hours of culture in the presence of PHA. Five concentrations of PHA ranging from undiluted stock PHA-P to a 1:25 dilution were used in all tests. Determination of an accurate mechanism for expression of the results was a major problem because of the intrinsic variability of the mitogen stimulation method. Maximum mean stimulation values always were selected, because stimulation profiles vary from individual to individual; e.g., one individual's cells may stimulate maximally at the highest mitogen concentration, whereas another's may do so at the lowest. In this series of experiments, the responses of cells from a normal individual and a cancer patient always were tested on the same day and compared. Because of JNCI, VOL. 63, NO.3,
Downloaded from https://academic.oup.com/jnci/article-abstract/63/3/587/1056406 by University of Durham user on 03 March 2018
SEPTEMBER 1979
590 Wood, Neff, and Stephens TABLE 2.-Contribution of T-cells. Bscells, and monocytes to mono-
TABLE 3.-Representative mitogen stimulation data on mononu-
nuclear cell suspensions derived from an unselected series of cancer patients
clear cells from cancer patients
T-lymphocytes"
B-lymphocytes b
2
33 18 32 40
4
44
4
23 63 21 16 28 48 35 64 58 64 51 48 53 53 60
63 52 69 60 51 71
