Altered monocyte function in uremia

CLINICAL

IMMUNOLOGY

AND

56, 66-80 (1990)

IMMUNOPATHOLOGY

Altered Monocyte ROCHELLE

A. S.

GIBBONS,* MARVIN

*Immunogenetics

Function in Uremia OLIVIA

R.

M.

MARTINEZ,?

AND

GAROVOY*

and Transplantation Laboratory and iLiver Transplantation Unit, Department of Surgery, University of California, San Francisco, California p4143

Uremia appears to suppress immune function predisposing patients to infections. the defect in cellular immunity was studied by exposing mononuclear cells (MNC) from uremic patients and controls to tetanus toxoid, diptheria toxoid, or Candida albicans antigen in vitro, the uremic cells were far less responsive. Monocytes and T cells, which are both involved in the proliferative response to soluble antigens, were isolated from MNC of uremic patients and HLA class II matched controls and incubated with tetanus toxoid. Tetanus toxoid-pulsed uremic monocytes were unable to stimulate the proliferation of HLA identical control T lymphocytes. Lymphocytes from uremic patients, however, were stimulated by tetanus toxoid-pulsed control monocytes. Therefore, the ability of monocytes to function as accessory cells is severely affected by uremia. The uremic monocytes were FcR+ , produced IL-lg, and expressed levels of HLA class II antigens comparable to controls. Although the biochemical defect in uremic monocytes remains unknown, the abnormality could explain many of the immunological changes of uremia. 0 19!30 Academic Press. Inc. When

INTRODUCTION

Uremic patients have an abnormal immune response which results in increased susceptibility to viral and bacterial infections (l-3). The immunological defect of these patients has been attributed to changes in both T and B cell function (4-6). Only a few reports concerning the effect of uremia on monocytes, a cell involved in host defense against microbes, have been published. Decreased phagocytosis (7), hydrogen peroxide production (8), and chemotaxis (9) have, however, been observed. This study shows that another activity of monocytes, accessory cell function, is also altered by uremia. Monocytes are a phenotypically and functionally heterogenous population whose subsets may be distinguished by the presence or absence of surface Fc receptors (FcR) (10, 11). Monocytes that bear HLA class II molecules and synthesize interleukin-ll3 (IL-lp) can act as accessory cells in elimination of antigens by lymphocytes (12). It has been suggested that these accessory cells constitute a subpopulation of monocytes that lack FcR (13). In this report we investigated the ability of uremic monocytes to respond to the antigens, tetanus toxoid, diptheria toxoid, and Candida afbicans, as determined by the proliferative response of T lymphocytes in vitro. We found that monocytes from the peripheral blood of patients with uremia were altered with respect to their function as antigen-presenting cells despite normal HLA class II expression and IL-ll3 synthesis. The possibility that the defect is due to an alteration in subsets of FcR+ and FcR- monocytes was examined. 66

0090-1229/90 $1.50 Copyright All ri&ts

8 1990 by Academic Press, Inc. of reproduction in any form reserved.

MONOCYTES

MATERIALS

IN

UREMIA

67

AND METHODS

Subjects

Citrated, venous blood was collected from hemodialysis patients who had endstage renal disease and who were undergoing evaluation for transplantation. All patients were drawn prior to dialysis. Characteristics of the patient population are shown in Table 1. Included in the study were 15 uremic females and 35 uremic males with a mean age of 41 years (range 12-74 years). The average duration of hemodialysis was 17 + 30 months (range = 1-166 months). The sex, age, duration of dialysis, or type of disorder did not appear to affect any in vitro parameter studied. Control blood samples were obtained from laboratory personnel or living related donors prior to surgery. Cells were serologically typed for HLA class I and II antigens following procedures specified by the American Society of Histocompatibility and Immunogenetics in the Immunogenetics and Transplantation Laboratory, UCSF. Mononuclear

Cell Isolation

The blood samples were diluted 1:2 with Hanks buffered saline lacking Mg*+ and Ca*’ (HBSS-CMF). Thirty milliliters of the mixture was underlayed with 10 ml of Ficoll-Hypaque (Pharmacia, Piscataway, NJ) and centrifuged for 20 min at ambient temperature at 400g. The peripheral blood mononuclear cells (MNC) at the interface were recovered, washed three times, and counted in a hemacytometer with ethidium bromide/acridine orange staining as an indicator of viability. Monocytes were isolated by a modification of the method of Ackerman and Douglas (14). Briefly, 10ml MNC (1 x 106/ml)in RPM1 1640containing 20% fetal bovine serum (FBS) were placed in 75cm* flasks coated with an exudate from P388Dl cells. The flasks were incubated for 1.5 hr at 37°C in an atmosphere of 5% CO2 in air and then the nonadherent cells were poured off, centrifuged, and counted. The adherent cells were washed two times with warm HBSS-CMF and incubated with 5 ml of phosphate-buffered saline containing 0.04% EDTA at 37°C for 10 min. The detached cells were harvested, irradiated (3000 tad), and diluted to 0.8 x 106/ml in RPM1 1640 containing 10% heat-inactivated normal human serum (NHS), henceforth referred to as complete medium. The cells recovered by this process were 800% monocytes as determined by foreward scatter on a fluorescent-activated cells sorter (FACS IV, Becton-Dickinson, Mountain View, CA) and were 80% Leu M3 + as assessedby cytofluorography . Anti-Leu M3 mAb recognizes 79-93% of normal human peripheral blood monocytes and does not react with lymphocytes, erythrocytes, or platelets (15). The percentage of monocytes from the MNC of uremics was notably higher (22.9 +- 3.7%) than that of the controls (13.5 + 1.8%). T lymphocytes were obtained by rosetting the nonadherent fraction of MNC with sheep red blood ells (SRC) treated with 2-aminoethylisothiouronium bromide (AET) (16). Rosetted cells contained less than 1% monocytes and were used in assaysfollowing lysis of AET-SRC with Tris-ammonium chloride buffer and three washes with HBSS-CMF.

68

GIBBONS,

MARTINEZ,

CHARACTERISTICS Uremic

No.

Age 24 21 67 43 32 40 29 51 43 45 60 22 34 49 42 20 14 22 39 25 52 26 54 52 43 60 42 36 66 35 31 36 52 37 35 52 31 52 53 31 40 60 69 31 38 12 36 50 50 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 31 38 39 40 41 42 43 44 45 46 41 48 49 50

AND

GAROVOY

TABLE 1 OF UREMIC

PATIENTS

Sex

(months)

M F M M M F M M M M M M M M M F M M F F F F F M M M M M F M F M M M M M M M M M F M F M M F F F M M

4 5 6 6 36 14 NA” NA 2 48 10 I 1 NA 1 1 NA 1 166 5 NA NA 12 12 18 1 12 1 5 14 5 14 5 24 Ni 46 72 2 NA 14 NA NA 33 3 4 3 NA 1

-

Disorder .___--~ Hereditary nephritis Hemolytic uremia Diabetes Diabetes Diabetes Chronic glomerulonephritis Chronic ~omendonephritis Ideopathic Nephrosclerosis F’yelonephritis Polycystic Focal sclerosing glomerulonephritis Chronic glomerulonephritis Chronic glomerulonephritis Ideopathic Obstructive uropathy Obstructed uropathy Focal sclerosing glomerulonephritis Chronic glomerulonephritis Other immune nephritis Polycystic disease ldeopathic Diabetes Ideopathic Nephrosclerosis Chronic glomerulonephritis Nephrosclerosis Chronic glomendonephritis Congenital urologic disease Hereditary nephritis Diabetes Hereditary nephritis Diabetes Diabetes Diabetes Membrane-proliferative glomerulonephritis Chronic glomeruloneptitis Chronic glomerubnephritis Focal sclerosing glomerulonephritis Ideopathic Lupus nephritis Diabetes Ideopathic Chronic glomerulonephritis Polycystic disease Congenital urologic disease Diabetes Diabetes Hereditary nephritis Chronic glomerulonephritis

a Not available.

All cell culture reagents were purchased from the University Culture Facility, San Francisco, California. Response

of MNC

MNC (1

x

of California

Cell

to Soluble Antigens

105/well) suspended in complete

medium

were placed in %-well

MONOCYTES

IN UREMIA

69

round-bottom microtiter wells for l-g days with 0,0.027,0.27, or 2.7 LFU tetanus toxoid or 0.013 LFU/ml diptheria toxoid (Massachusetts Biological Laboratory, Jamaica Plains, MA) in a total volume of 0.2 ml. In one experiment, C. albicans antigen (a gift from Dr. Dewey Moody, Applied Immune Sciences, Menlo Park, CA) was diluted in complete medium to a final concentration of 50 ug/ml which had been found to produce maximal stimulation of MNC. Proliferation was measured as described below. Antigen-Presenting

Function

of Monocytes

The ability of isolated monocytes to present tetanus toxoid to T lymphocytes was tested by placing lO?ml monocytes in 15ml round-bottomed tubes, irradiating (3000 rad) the cells, and incubating them with or without 0.27 LFU/ml tetanus toxoid in complete medium for 18 hr at 37°C. Monocytes were recovered by placing the tube on ice for 15 min, vortexing, and washing three times with HBSS-CMF to remove unbound tetanus toxoid. Tetanus toxoid-pulsed monocytes (2 x 104)were placed into wells of microtiter plates with 1 x 10’ T cells from either control or uremic individuals in a final volume of 0.2 ml. Cultures were incubated at 37°C in an atmosphere of 5% CO, for 6 days. The response of the T cells to the tetanus toxoid-pulsed monocytes was determined by measuring the extent of proliferation as described below. Proliferation

Studies

DNA synthesis was assayedby adding 1 &i/well tritiated methyl thymidine (sp act 6.7 Ci/mM; New England Nuclear Corp., Boston, MA) to the microtiter wells and incubating for lg-21 hr. The incorporation of tritiated methyl thymidine was determined after harvesting by liquid scintillation counting (Beckman Instruments, Irvine, CA). The results were expressed either as counts per minute (cpm) or as a stimulation index (SI) that was calculated as in Eq. [l]: SI = cpm of cells incubated with tetanus toxoid/ cpm of cells incubated without tetanus toxoid.

ill

IL-1 p Production

MNC were obtained as described above. The percentage of monocytes was estimated by foreward scatter characteristics. The MNC were diluted to 1 x lo6 monocytes/ml in complete medium and 0.2 ml of the suspension was placed in wells of a flat-bottomed %-well microtiter plate. The cells were incubated for 2 hr at 37”C, and the nonadherent cells were removed by washing three times with warm RPM1 1640. Then, 0.2 ml of RPM1 was added to the wells with O-O.1 pg/ml lipopolysaccharide (LPS) (Difco, Detroit, MI). After 20 hr, supematants were collected and stored at -70°C until assayed for IL-ll3 by ELISA (Cistron, Pine Brook, NJ).

70

GIBBONS,

Determination

MARTINEZ,

AND

GAROVOY

of HLA Class II and Fc Receptor Expression

by Monocytes

HLA class II expression by monocytes was analyzed by flow cytometry. MNC ( 106) were double stained according to the manufacturer’s recommendations with the phycoerythrin (PE) and fluorescein isothiocyanate (FITC)-coupled monoclonal antibodies (mAbs) of interest (Becton-Dickinson). Monocytes were identified by staining with anti-Leu M3 mAbs conjugated to PE, and HLA class II antigen expression was detected by staining with anti-HLA-DR. -DQ, or -DP mAbs that were conjugated to FITC. To evaluate FcR expression by monocytes, the cells were double stained with anti-Leu M3 mAb conjugated with PE and either anti-FcR (I)-FITC or anti-FcR (II)-FITC mAbs that were obtained from Medarex (West Lebanon, NH). Background was determined by staining with FITC- or PE-labeled isotype matched, nonspecific mAbs. Analysis of the results was performed on a Becton-Dickinson research analyzer with Consort 30 software. Using anti-Leu M3 mAbs to identify monocytes, a gate was established using volume and side scatter parameters. No difference in scatter patterns was observed between monocytes obtained from patients and controls. Data are presented as dual parameter histograms on a logarithmic scale. Mean channel fluorescence (MCF) was calculated for each population of cells stained with the fluorescent mAb of interest (16). Calibration of the instrument established that a MCF difference of 25 channels on the log scale represented a twofold change in fluorescence. MCF values were used to correlate fluorescence to relative antigen density between samples. The percentage of monocytes positive for an HLA class II antigen (DR, DQ, DP) or FcR was calculated in Eqs. [2] and [3], respectively: % Class II+ monocytes

= Leu-M3+

Class II+ cells x lOO/all Leu M3+ cells PI

% FcR+ monocytes Background stained cells. Statistical

= Leu-M3+

FcR+ cells x lOO/all Leu M3+ cells.

[3]

staining (~1%) was subtracted from the percentage of positively

Methods

The results were expressed as the mean of triplicate or hexicate wells 2 the standard deviation. Differences between group means were analyzed with a Student’s t test. RESULTS Response of Uremic MNC to Tetanus Toxoid Figure 1 shows the 6-day proliferative response to 0.27 LFU/ml tetanus toxoid of MNC from the controls and uremics (Patients l-48, Table 1). The magnitude of the response to tetanus toxoid was highly variable in the 43 control cultures (SI = 46 + 33, range = 6-147). Nevertheless, the mean SI of the response of 48 uremic individuals was much lower (SI = 4 2 5, range = 0.5-17.6) when compared to the

MONOCYTES

IN

71

UREMIA

2 1

-moo.....

1J z

Uremic

FIG. 1. The response of MNC from controls and uremics to tetanus toxoid. MNC were incubated for 5 days with or without 0.27 LFU/ml tetanus toxoid and proliferation rates were then determined as described under Materials and Methods. The results, which are the average of hexicate cultures, are shown as a stimulation index.

mean SI of the controls. The difference between the mean SI of the controls and uremics was highly significant (P < 0.001). These data show that MNC from uremics have a markedly diminished proliferative response to tetanus toxoid as compared to the MNC of controls. Figure 2 demonstrates the proliferative response of MNC from two controls and three uremic patients (Uremic 40, 41, and 42, Table 1) to 0, 0.027, 0.27, and 2.7 LFU/ml tetanus toxoid. MNC of the controls and uremic patients cultured without tetanus toxoid synthesized DNA at low levels. The addition of 0.027 LFU/ml or 0.27 LFU/ml of tetanus toxoid to cultures of MNC increased DNA synthesis 0 Control w Control q Urembc-40 Uremfc-41

n

0.0

0.027

0.27

Uremic-42

2.7

Tetanus Toxoid (LFUlml)

FIG. 2. The response of MNC from controls and urernics to various concentrations of tetanus toxoid. MNC were incubated for 5 days with 0, 0.027, 0.27, or 2.7 LFUlml tetanus toxoid and proliferation rates were then determined as described under Materials and Methods. The results, which are the average of triplicate cultures f the standard deviation, are shown.

12

GIBBONS,

MARTINEZ,

AND

GAROVOY

rates of the control cultures dramatically. MNC from one uremic patient (Uremic 40) proliferated slightly in response to tetanus toxoid, but the MNC from two other uremic individuals did not. Concentrations of 2.7 LFU/ml were toxic to the cultures of both controls and uremic MNC. The results demonstrated that the proliferative response of MNC from patients with uremia to tetanus toxoid is either much lower or absent compared to that of controls over a broad concentration range. Kinetics

of MNC

Response

to Tetanus

Toxoid

To determine the kinetic profile of the proliferative response of normal and uremic (Uremics 43, 44, and 45, Table 1) MNC to tetanus toxoid, replicate cultures of MNC were incubated with or without 0.27 LFU/ml tetanus toxoid (Fig. 3). Proliferation rates were determined on days l-8 of culture. The rate of incorporation of i3H]TdR into DNA of the MNC from the normal individuals increased on each successive day until days 5 and 6. After days 6 and 7 the proliferative response declined dramatically. The cultures from two of the uremic patients showed no significant proliferation throughout the study. A third patient (Uremic 43), with a low response to tetanus toxoid, showed an increased rate of proliferation until Day 5. Therefore, the abnormal response to tetanus toxoid of uremic cells was not due to altered kinetics. Stimulation of Lymphocytes Individuals and Controls

by Tetanus Toxoid-Pulsed

Monocytes

from

Uremic

In order to assess monocyte and T cell function in the tetanus toxoid proliferation assay, isolated monocyte and T cell fractions were obtained from MNC of uremic patients (Uremic 49 and 50, Table 1) and controls. Monocytes from the control or uremic patients, that were incubated in medium alone, stimulated the 150,

M

Control Control

-

Uremsc-43

-

Uremc-44

-

Uremic-45

0 0123456789 Day

FIG. 3. Kinetics of response to tetanus toxoid. MNC from two normal individuals (0,O) and three uremic patients (A, A, n ) were incubated with or without 0.27 LFU/ml tetanus toxoid for l-8 days. On each day, proliferation rates were determined as described under Materials and Methods. The results are the average of triplicate cultures and are shown as a stimulation index. The standard deviation was ~15% for all values.

MONOCYTES

73

IN UREMIA

proliferation of cultures of T lymphocytes from the control or uremic individuals slightly (Table 2A). Table 2B shows the results of pulsing monocytes from control individuals with tetanus toxoid for 18 hr, washing them to remove unbound tetanus toxoid, and then culturing them with autologous T lymphocytes or HLA identical T lymphocytes from uremic patients. The purified T lymphocytes from both controls and uremic patients proliferated vigorously in response to the tetanus toxoid-pulsed monocytes from the control individuals. When tetanus toxoidpulsed monocytes from uremic patients were cultured with control or uremic T cells the proliferation was significantly lower than the response to tetanus toxoidpulsed monocytes from control individuals (P < 0.001). These results show that the altered proliferation of MNC from uremic patients to tetanus toxoid is due to a defect in monocyte function. Response of MNC from Uremic and Controls to Diptheria C. albicans

Toxoid and

Table 3 shows the results of exposing MNC from uremics (Uremic 40,41, and 42, Table 1) and controls to 0.013 LFU/ml of diptheria toxoid. MNC from controls showed increased DNA synthesis in response to the antigen. Uremic 40, who could respond to stimulation by tetanus toxoid (Fig. 2), was also responsive to diptheria toxoid, although both responses were lower than the controls. The two other uremic individuals (Uremic 41 and 42) had low or absent stimulation indices to both tetanus toxoid and diptheria toxoid (Fig. 2 and Table 3, respectively). Table 4 compares the effect of 50 pg/ml of C. albicans and 0.27 LFU/ml tetanus toxoid on MNC from three uremics (Uremic 46,47, and 48, Table 1). The cultures of MNC from two controls and one uremic (Uremic 46) were stimulated by tetTABLE 2 RESP~NSEOFCONTROLANDURJNICTLYMPHOCYTESTOSTIMULATION TOXOID-PULSED MON~CYTES

BYTETANUS

T lymphocytes [3H]TdR incorporation (cpm) Control A. Monocytes incubated with medium alone Experiment 1 Control Uremic Experiment 2 Control Uremic B. Tetanus toxoid-pulsed monocytes Experiment 1 Control Uremic Experiment 2 Control Uremic

3,551 5 1253 1,547 + 271 745 2 80 959 f 231

Uremic

1,281 ” 335 599 + 247 440 + 31 402 + 115

18,791 + 6254 1,893 2 523

51,%7 f 1592 855 f 68

23,597 2 1592 2,573 2 593

18,661 k 2436 1,501 k 142

a The results, which are the average of triplicate cultures, show the counts per minute of tritiated thymidine incorporated into DNA of cells k the standard deviation.

74

GIBBONS,

MARTINEZ, TABLE

COMPARISON

AND GAROVOY 3 MNC TO STIMULATION

OF RESPONSE OF CONTROL AND UREMIC DIPTHERIA TOXOID”

Medium

- ~--.. Control Control Uremic 40 Uremic 41 Uremic 42

1120 324 396 425 300

BY

Diptheria toxoid

2 562 f 64l 4 88 I? 32 2 130

13,586 2 2341 16,265 2 13% 2,506 +- 13% 701 2 36 197 -+ 116

0 The results, which are averages of triplicate cultures, show the counts per minute of tritriated thymidine incorporated into the DNA of the cells 2 the standard deviation.

anus toxoid. C. albicans antigen stimulated all cultures to a lesser extent than did tetanus toxoid. The three uremic individuals again had a lower response than did the two controls. These results demonstrated that MNC from uremic individuals exhibit a decreased proliferative response to several soluble antigens. Additionally, patients with low responses to tetanus toxoid also responded only slightly to other soluble antigens. Those individuals who did not respond to tetanus toxoid were unable to respond to diptheria toxoid or C. albicans antigen. A general defect in the ability of monocytes to present soluble antigen is present in uremic patients. MHC Class II Expression by Uremic Monocytes

The ability of monocytes to function as accessory cells in T cell proliferative responses to soluble antigen requires expression of HLA class II molecules (12). Table 5 compares the expression of HLA class II molecules on the monocytes of uremic individuals and controls. Monocytes were double-stained with anti-Leu M3 mAb and mAbs for the HLA class II molecules, DR, DQ, and DP. The percentage of normal monocytes bearing both Leu M3 and HLA-DR, DQ, or DP was 73 f 14%, 20 + 8% and 21 k 7%, respectively. The monocyte populations from the uremic individuals showed similar expression of Leu M3 and HLA class II molecules (DR = 71 2 14%, DQ = 19 2 13%, and DP = 27 + 6%) as compared to controls. TABLE COMPARISON

OF RESFVNSE

4

OF CONTROL AND UREMIC TOXOID AND Candida

MNC

TO STIMULATION

[‘H]TdR incorporation Medium Control Control Uremic 46 Uremic 47 Uremic 48

1734 1012 1809 942 1561

+ a k * f

580 219 820 136 688

BY TETANUS

albicans

Tetanus toxoid 101,550 90,308 54,639 806 1,420

2 2 f + +

9,540 11,610 11,082 425 74

@pm) C. albicans 11,266 6,034 4,096 1,434 2,388

f + + k k

4136 1294 220 453 84

a The results, which are averages of triplicate cultures, show the counts per minute of tritriated thymidine incorporated into the DNA of the cells 2 the standard deviation.

MONOCYTES TABLE HLA CLASS II ANTIGEN

-___

75

IN UREMIA 5

EXPRESSION

BY

Leu M3 + MONOCYTES

Control

Uremic

HLA

n

% Positive cells”

MCFb

n

% Positive cells’

MCFb

DR

25 23 9

73 + 14 20 e 8 21 *7

132 2 36 116 * 36 112 IT 48

14 14 7

71 2 14 19 * 13 27 f 6

144 k 16 132 + 20 132 k 12

DQ -

DP

Note. Mononuclear cells were stained with anti-Leu M3 mAb and anti-HLA-DR, -DQ, or -DP mAbs and analyzed with a FACS research analyzer with Consort 30 software. u Percentage of monocytes that were positive for both Leu M3 and HLA-DR, -DQ, or -DP. ’ MCF, an estimate of the relative density of antigens.

The MCF is an estimate of the relative density of HLA-DR, -DQ, and -DP antigens expressed by the Leu M3 + cells of controls and uremics. It was found that the density of HLA-DR, DQ, and DP of the Leu M3 + cells from the uremic individuals was slightly greater than that of controls but the differences were insignificant (Table 5). Therefore, the defective response of the uremic monocytes to tetanus toxoid was not due to inadequate expression of HLA class II molecules . Expression tif FcR FcR - monocytes may constitute the antigen presenting subset of monocytes. To determine if the monocyte population from uremic patients was altered with respect to the percentage of cells that expressed FcR, cells were double stained with anti-Leu MZPE mAbs and with FITC-conjugated mAbs to FcR (I) and FcR (II). As shown in Table 6, greater than 70% of the uremic and control monocytes were positive for both Leu M3 and for FcR (I) and FcR (II). The percentages of cells that double stained with both anti-Leu M3 mAb and either anti-FcR (I) or FcR (II) mAbs were comparable in controls and uremics.

EXPRESSION

OF

FcR BY PERIPHERAL Leu M3 + FcR +

Control

Uremic

FcR (I) FcR (II)

73 * 8 74 k 8

74 f 8 79 k 7

FcR (I) FcR (II)

11628 148 t 4

136 2 16 148 -+ 4

TABLE 6 BL~~D MONOCYTES

FROM

UREMIC

Leu M3 + FcR Control

Uremic

% Positive cells” 0.4 2 0.2 0.5 t 0.5 0.1 * 0.1 0 MCFb 60 t 0.4 0

36 f 48 0

PATIENTS

AND CONTROLS

Leu M3 - FcR + Control

Uremic

12.8 r+ 0.9 11.2 f 2

12 -r- 5 14 2 4

116 f 8 132 2 4

116 f 16 136 -t 4

Note. Mononuclear cells were stained with anti-Leu M3 mAb and either anti-FcR (I) or antiFcR (II) mAbs and analyzed with a FACS research analyzer with Consort 30 software. a Percentage of Leu M3 + cells that were positive for either FcR (I) or FcR (II). b MCF, the relative antigen density.

76

GIBBONS,

MARTINEZ.

AND

GAROVOY

In both controls and uremics a population of monocytes that were Leu M3-FcR (I) + and a population that was Leu M3-FcR (II) + existed. Less than 1% of the cells from the controls and uremics that stained with anti Leu M3 mAb were FcR (I) or FcR (II) negative. The MCF for FCR (I) and FCR (II) by monocytes from uremic and control populations was equivalent, which indicated that the relative density of the antigens on the surface of the monocytes was not altered by uremia. The results demonstrated that monocytes from control and uremic patients have similar distributions of FcR (I) and FcR (II). Furthermore, there was no indication of a significant population of FcR- monocytes in human peripheral blood. Synthesis

of IL-l f3 by Uremic

and Control

Adherent

Cells

Activation of resting T cells requires multiple monocyte-derived signals including production of IL-lp. Exogenous IL-ll3 did not restore the defective proliferative response of uremic monocytes (data not shown). To assess the ability of monocytes to produce IL-l& adherent cells obtained from MNC of uremics and controls were cultured with LPS for 24 hr, supernatants harvested, and assayed for IL-lp. Table 7 shows the amount of IL-lp secreted into the medium of the cultures as determined by ELBA. It was demonstrated that the amount of soluble IL-lp produced in response to LPS was comparable in both groups. DISCUSSION

We have shown that MNC isolated from uremic patients have a reduced or absent response to the soluble antigens, tetanus toxoid, diptheria toxoid, and C. albicans, as determined by the extent of proliferation of T lymphocytes. The reaction of immune cells to soluble antigen requires a complex series of events involving both monocytes and T cells. Monocytes internalize and digest the antigen, which is reexpressed on the surface of the cell in association with HLA class II molecules. Specific T lymphocytes then respond to the altered surface of the monocyte by proliferating. To determine what cell population in the MNC was defective in responding to tetanus toxoid, monocytes and T cell populations were isolated from HLA identical normal and uremic individuals. Normal and uremic monocytes were pulsed with tetanus toxoid, washed, and added back to either autologous or HLA identical donor T lymphocytes. The degree of stimulation by the monocytes was TABLE LPS INDUCED

Control Uremic

SECRETION

OF IL-lp

I

BY CONTROL

AND UREMIC

MON~IZYTES

IN

VITRO

n

IL- 1p (pg/ml)

5 7

7005 + 1909 8819 t 1221

Note. Adherent peripheral blood cells were cultured for 20 hr in RPMI 1640 containing 0.1 &ml LPS. Supematants were harvested and the amount of IL-lg was determined by ELISA. Results show the average of hexicate wells 2 the standard deviation.

MONOCYTES

IN

UREMIA

77

determined by measuring the rate of proliferation of T lymphocytes. Uremic monocytes were not able to stimulate significant proliferation of HLA identical normal T cells. In contrast, normal monocytes were able to activate the uremic T cells. The results, therefore, suggested that the failure of uremic MNC to respond to tetanus toxoid was due to altered monocyte function. The defect in the uremic monocytes was apparent over a broad range of concentrations of tetanus toxoid and decreased reactivity of uremic MNC to other antigens, such as diptheria toxoid and C. afbicans, was also observed. The monocyte lesion was not due to decreased HLA class II expression or reduced secretion of IL-ll3 as assessedby response to LPS in vitro. Reduced responses to soluble antigens have been reported in a variety of conditions. MNC from patients with injuries from burns or accidental or surgical trauma have low proliferative responses to tetanus toxoid (17, 18). Additionally, monocytes from neonatal mice and humans have reduced antigen-presenting capabilities (19). In all these situations, concomitant reductions in class II expression, which is required for antigen presentation (12), have been described and could explain the abnormalities. Other defects have been reported in patients with altered responses to soluble antigens. Individuals with lepromatous leprosy do not respond to stimulation by Mycobacterium leprae due to a T cell defect (20). Decreased production of cytokines by cells may also be involved in abnormal responses to antigens. Patients with pulmonary tuberculosis, for example, have a tuberculin-specific defect in IL-2 production (21). In other patients, hypoimmunity has been ascribed to alterations in subpopulations of monocytes (13). Peripheral blood monocytes consist of cells with distinct immunoregulatory functions. Monocyte subsets, which can be isolated on the basis of expression of Fc receptors, appear to have different capacities to synthesize IL-ll3 and release PGEz (22). It has been proposed that antigenpresenting monocytes constitute a subset of cells negative with respect to FcR expression (13). Separation into FcR + and FcR - subpopulations has relied on the ability of monocytes to adhere to human red cells coated with IgG (10, 11). Since 75-95% of the monocytes bind to red cells (10, 1l), the FcR - population would then consist of 5 to 25% of the monocytes. Fluoresceinated mAbs to both the high affinity FcR (I) and low affinity FcR (II) have recently become available and provide a direct and quantitative method to define FcR + and FcR - populations (see Materials and Methods). These reagents were used to determine whether differences in the distribution of FcR+ and FcR- cells occurred in peripheral blood monocytes from uremic patients and controls. Most monocytes expressed Leu M3, FcR (I), and FcR (II). A small percentage (< 1%) of the Leu M3 + cells did not possess FcR (I) or FcR (II) in both uremic and control monocyte populations. Ten to 15% of the monocytes from controls and uremics did not express Leu M3, but were positive for FcR (I) or FcR (II). The density of the receptors on the cell surface of monocytes from the controls and uremic patients was also comparable. The results suggest that human monocyte populations could not be divided into subpopulations based

78

GIBBONS,

MARTINEZ,

AND

GAROVOY

upon the expression of FcR and that cells from uremic individuals did not differ from the controls with respect to expression of FcR. The defect in antigen processing by monocytes shown here may explain the humoral immunological alterations observed in uremic patients both in vitro and in viva. The synthesis of antibody by B lymphocytes is the culmination of a cascade of events that is initiated by the processing of antigen by accessory cells. In this regard, B lymphocytes from uremic individuals have been shown to produce less IgG in response to pokeweed mitogen than controls in vitro (6). Uremic patients also may not respond well to vaccination as determined by serum antibody titer after immunization. Patients on hemodialysis with end-stage renal disease have been shown to synthesize lower amounts of antibody in response to immunization with hepatitis B antigen than control individuals (23). In the former study, 50% of the male uremics and 34% of female uremics did not produce any detectable antibody as a response to immunization with hepatitis B antigen a year after beginning treatment (23). Lower serum antibody titers have also been noted in patients undergoing hemodialysis that were immunized with inlluenza virus when compared to normal persons (24). Other workers, however, have not found abnormal responses to immunization with pneumococcal vaccination by hemodialysis patients unless they also had chronic renal failure (25). Nevertheless, the effect of uremia on the efficiency of vaccination of uremic patients is unclear. Some workers have reported that serum antibody titers of hemodialysis patients with chronic renal disease were similar to the control populations after immunization with influenza virus (26). In this paper we have not addressed the intracellular events which occur during antigen processing. Changes in blood products that result from uremia may affect one of the steps involved. We have found that normal monocytes incubated with uremic serum have a decreased ability to function as antigen-presenting cells (manuscript in preparation). Further studies on the defect of uremic patients’ monocytes, with respect to antigen presentation, are being undertaken to elucidate the biochemical lesion involved. ACKNOWLEDGMENTS The authors are grateful to Mr. Victor L. Lim for his help with flow cytometry, to Mr. Calvin Lou for assistance with clinical data, to Ms. Mary Math for help in preparing the manuscript, and to Drs. Noel Warner and Anne Jackson of Becton-Dickinson for providing many of the fluoresceinated monoclonal antibodies.

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