JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1989, p. 1748-17530095-1137/89/081748-06$02.00/0Copyright 1989. American Society for Microbiology

Effect of Monophosphoryl Lipid A on the In Vitro Function ofPeritoneal Leukocytes from Uremic Patients on Continuous

Ambulatory Peritoneal DialysisSILVIA CAROZZI,' MARC SALIT,2* ALBERTO CANTALUPPI,2 MARIA GRAZIA NASINI, SERGIO BAROCCI,3

SEBASTIANA CANTARELLA,3 AND SILVANO LAMPERI4

Nephrology and Dialysis Unit, St. Paul's Hospital, Sai'onca,1 and Nephrology and Dialyvsis Departinent4' and ininunologyDepartmentt3 St. Martin's Hospitcal, Genou, Ital', and Baxter Healthcare Corp., Rollnd Lake, Illinois 600732

Received 21 February 1989/Accepted 17 April 1989

We analyzed the effects of monophosphoryl lipid A (MPL), a relatively nontoxic immunostimulant derivedfrom bacterial endotoxin, on the depressed in vitro immune function of leukocytes derived from six patientsundergoing continuous ambulatory peritoneal dialysis and who had histories of recurrent bacterial peritonitis.MPL was also tested for its capacity to stimulate the proliferation of peritoneal fibroblasts, as determined by[3H]thymidine incorporation. In vitro incubation of peritoneal lymphocytes and macrophages (PM+) withincreasing amounts of MPL, up to 5 ,ug/ml, resulted in a dose-dependent enhancement of gamma interferonand interleukin-2 production by peritoneal lymphocytes and interleukin-1 release by PMq. In vitro incubationof PM4 with MPL also resulted in an increase of PMc bacterial killing and membrane Fc receptor number,although no change in peritoneal fibroblast proliferation was seen with any of the MPL concentrations tested.These results suggest that the peritoneal leukocyte dysfunction observed in patients undergoing continuousambulatory peritoneal dialysis and who have high rates of peritonitis may be alleviated, to some degree, byMPL, without directly inducing a potentially deleterious fibrotic lesion.

Despite improvements in treatment techniques and equip-ment, bacterial peritonitis still remains one of the majorcomplications of continuous ambulatory peritoneal dialysis(CAPD) (K. D. Nolph, Proceedings of the 8th NationalConjerence on CAPD, in press); however, epidemiologicaldata indicate that peritonitis is not randomly distributed.Some patients (about 20% of the total CAPD patient popu-lation) develop bacterial peritonitis more often than others,and studies have shown that patients with high infectionrates have abnormalities of peritoneal cellular and humoralimmune defense mechanisms which lead to defective bacte-ricidal activity. Such dysfunctions seem to play a primaryrole in the pathogenesis of relapsing peritoneal infections(14, 16).Monophosphoryl lipid A (MPL), a substance that is ob-

tained by acid hydrolysis of bacterial endotoxin, has beenshown to induce immunostimulatory effects such as cytokineproduction, spleen cell mitogenesis, macrophage (PM4)activation, and a variety of other effects on the cell-mediatedimmune response (20, 21, 24-26).

Consequently, we wanted to evaluate the in vitro effects ofvarious concentrations of MPL on the function of peritonealcells lymphocytess and PMb) taken from CAPD patientswho were selected from a cohort with a high incidence ofperitonitis.

Since previous research has demonstrated that a numberof immunoregulatory molecules are able to induce fibroblastproliferation (which is an undesirable side effect of immu-nostimulation), the in vitro effects of MPL on the prolifera-tion of peritoneal fibroblasts were also studied (2, 5).

MATERIALS AND METHODS

Patient population. Six uremic patients who had beenundergoing CAPD for 37 to 56 months (mean, 44 + 6.6

Corresponding author.

months) were studied for their reactivities to MPL. Thecriterion for selection was an overall peritonitis incidence ofone episode of peritonitis per 8 patient-months or more

(mean, one episode per 7.2 ± 0.5 patient-months). Threemales and three females (ages, 37 to 61 years; mean, 49.5 ±

7.9 years) were included in the study. Primary renal diseaseswere chronic glomerulonephritis (two cases), chronic pyelo-nephritis (two cases), and malignant nephrosclerosis andpolycystic disease (one case each). CAPD (Y-set) was theinitial and only therapy for these patients with end-stagerenal disease.A low-peritonitis-rate control group was composed of

eight CAPD patients who had never experienced peritonitisand were comparable for mean age, sex, and dialysis sched-ule to those with relapsing peritonitis.The dialysis schedule was four daily exchanges of a 2-liter

dialysate (Dianeal; Baxter Healthcare, Deerfield, 111.) withglucose concentrations ranging from 1.36 to 2.27 g/dl.

All patients in this study were free of symptoms ofperitonitis for at least 1 month prior to cell collection, and forat least 1 year prior to enrollment in this study no patient wasvaccinated nor was any patient treated with immunoglob-ulins, steroids, or immunosuppressive or immunostimula-tory drugs.Twenty healthy women undergoing laparoscopy for diag-

nostic evaluation of infertility constituted the source ofcontrol peritoneal lymphocytes (PLy) and PM4. None ofthese controls had clinical or laparoscopic evidence ofintraabdominal or intrapelvic inflammation or endometrio-sis.

Isolation of PLy and PMP. Immediately after peritonealdialysis effluent drainage, the entire overnight dialysateeffluent volume (ranging from 1,780 to 2,320 ml) of patientswith high peritonitis rates was centrifuged (500 x g) for 20min. Excess fluid was removed with a plasma extractor; a

1-liter portion of effluent yielded an average of 2.3 x 10' cells

1748

Vol. 27, No. 8

on August 25, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

MPL AND PERITONEAL IMMUNITY 1749

(range, 1.3 x 106 to 3.9 x 106 cells). Final cell suspensionshad a mean composition of 86%7 PM+, 9%c PLy, 3% neutro-phils, and 2%, eosinophils. Similar results were obtained forthe cells derived from the low-peritonitis-rate patients. Sam-ples containing more than 5% neutrophils were not includedin this study. PM4 were identified by Wright-Giemsa stain-ing and by nonspecific esterase (Sigma Chemical Co., St.Louis, Mo.) staining. PLy were defined by Wright-Giemsastaining and by the absence of homogeneous esterase stain-ing. Neutrophils were defined by Wright-Giemsa staining.PMq were separated from unfractionated peritoneal cell

suspensions by their adherence to plastic petri dishes (100 by15 mm; Falcon; Becton-Dickinson, Grenoble, France)coated with fetal bovine serum (FBS; GIBCO Laboratories,Grand Island, N.Y.) (1). Adherent cells were recovered bygentle scraping with a rubber policeman and were found tobe more than 95% PM4. The nonadherent peritoneal cellpopulation was depleted to less than 3 and 1% PM4> byrepeating the procedure two and three times, respectively.PMX were washed three times in RPMI 1640 medium(GIBCO) and suspended in RPMI 1640 medium or in Hanksbalanced salt solution with 0.1% gelatin at concentrationsranging from 1 x 106 to 5 x 106 cells per ml. More than 95%PM4 were viable by trypan blue exclusion.PLy were obtained from unfractionated peritoneal cell

suspensions by three consecutive 90-min adherence proce-dures in plastic petri dishes. Only cells obtained by gentleaspiration of the supernatant cell suspension into a pipettewere used. Nonadherent peritoneal cells were more than98% PLy, less than 1% PM4, and less than 1% neutrophils.PLy suspensions were washed three times in RPMI 1640medium and suspended in the same medium at a finalconcentration of 106 cells per ml.Normal peritoneal cells were obtained from healthy

women immediately after placement of the laparoscope andprior to surgical manipulations. Peritoneal fluid (44 + 15 ml;range, 27 to 64 ml), which was aspirated by manual suctionfrom the posterior cul-de-sac, averaged 6.6 x 105 cells(range, 4 x 105 to Il x 105 cells). Final cell suspensions weremade up of PM4~(93%), PLy (3%), neutrophils (2%), andeosinophils (2%). Cells were defined by their morphologicalfeatures on Wright-Giemsa staining. PMX stained positivelywith nonspecific esterase. More than 95% PMX were viableby trypan blue exclusion. Contaminating erythrocytes wereremoved by hypotonic lysis.To obtain purified normal PM4 and PLy, peritoneal cell

suspensions were processed in the same way as peritonealcells from CAPD patients were.

General experimental design. Purified populations of PMXor PLy from high-peritonitis-rate CAPD patients were incu-bated in vitro with different amounts of MPL. Then, culturesupernatants were tested for cytokines (gamma interferon[IFN-y], interleukin-2 [IL-2], interleukin-1 [IL-1i), andMPL-treated PMX were tested for Fc receptor density andbactericidal activity. Similarly prepared laparoscopy patient-derived peritoneal leukocytes or leukocytes obtained fromCAPD patients without a history of peritonitis were incu-bated with known stimulants of lymphocyte and macrophagefunction and assayed similarly.

Activation of PLy and PM4 with MPL. Before the assay ofIFN-y, IL-2, and IL-1 release, membrane Fc receptors,bactericidal capacity, and fibroblast proliferation, PLy,PMb, and peritoneal fibroblasts (suspended in RPMI 1640medium supplemented with heat-inactivated 10% FBS) wereincubated for 48 h at 37°C with concentrations of MPL (RibiImmunoChem Research Inc., Hamilton, Mont.) ranging

from 0.05 to 50 ptg/ml. Lyophilized MPL was diluted in watercontaining 0.2% triethylamine (TEA).As a negative control, peritoneal cells were incubated with

0.2%c TEA in water (no MPL) and with RPMI 1640 mediumalone.

Control production of IFN-y by PLy. A total of 106laparoscopy-derived or low-peritonitis-rate CAPD patient-derived PLy per ml in RPMI 1640 medium supplementedwith 10% FBS-10% 200 nM L-glutamine-10 - M 2-mercap-toethanol-100 IU of penicillin per ml-0. 1 mg of streptomycinper ml were incubated at 37°C for 3 days in a 5% CO,atmosphere with 10 Ftg of concanavalin A (Sigma) per ml(13).

Assay of IFN-y production by PLy. The amount of IFN-y inthe culture medium of PLy from CAPD patients treated withMPL or control cells treated with concanavalin A wasdetermined with an assay kit (IMRX; Centocor Corp.,Malvern, Pa.).Samples were incubated with polystyrene beads coated

with a mouse monoclonal antibody specific for human IFN-y; then, they were washed and incubated with a 1-51-labeledsecond mouse monoclonal anti-human IFN-y antibody di-rected to an epitope that was distinct from that recognizedby the first monoclonal antibody. The amount of IFN-y inthe supernatant preparation, expressed in units per milliliter,was estimated by comparison with a standard curve gener-ated by using purified IFN-y. The lower limit of detectionwas 0.1 U/ml.

Control production of IL-2 by PLy. Control cell IL-2 wasproduced by incubating 106 laparoscopy patient-derived PLycells or low-peritonitis-rate CAPD patient-derived PLy cellsper ml with 1 Ftg of phytohemagglutinin (PHA-P; DifcoLaboratories, Detroit, Mich.) per ml in RPMI 1640 mediumcontaining 10% normal human serum for 48 h at 37°C in a SYCO, humidified atmosphere. The cell-free supernatant washarvested after centrifugation of the culture for 10 min at 500x g, filtered through a 0.24->tm-pore-size filter, and stored at-20°C until it was assayed. Standard IL-2 was obtained fromhuman spleen lymphocytes that were adjusted to 5 x 106cells per ml in RPMI 1640 medium supplemented with 2%normal human serum and incubated with 1 ,ug of PHA-P perml for 48 h at 37°C in a 5Sc CO, humidified atmosphere. Thissupernatant was used both as a T-cell growth factor for thepreparation of a cultured cell line and as a reference prepa-ration for the titration of IL-2.

IL-2 assay. Activated human T cells were obtained byculturing peripheral blood lymphocytes from a normal do-nor, adjusted to 0.3 x 106 cells per ml, and incubated inRPMI 1640 medium containing 20% FBS and 25% standardIL-2 supernatant (7). Every 48 h the medium was changed,until day 14, when the cells were washed in IL-2-freemedium and suspended in RPMI 1640 containing 20% FBS.They were incubated for 2 days at 37°C to elute any absorbedIL-2 and then were washed and frozen in liquid nitrogen.These cells were then used in a microtitration assay ofIL-2-containing supernatants. IL-2 activity in the culturesupernatant of MPL- or PHA-P-treated PLy was measuredby the ability of the supernatants to induce mitogenic activ-ity in 2 x I04 activated T cells in a 72-h [3H]thymidineincorporation assay. Supernatants were tested in successivelog1 dilutions, and IL-2 units were expressed with referenceto the standard IL-2 preparation containing a known numberof units. Calculations were performed by probit analysis.

Control production and assay of IL-I. IL-1 production byPM> was determined by the method described by Gery et al.(6). Monolayers of 2 x 106 PMX were incubated in 1 ml of

VOL. 217. 1989

on August 25, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

1750 CAROZZI ET AL.

RPMI 1640 medium containing 5% FBS and 10 Fg ofbacterial lipopolysaccharide W from Salmonella typhimu-rium (Difco). Following 24 h of incubation, supernatantsfrom the PMX were tested for IL-1 activity in a thymocyteproliferation assay (capacity of supernatants to potentiatethe proliferative response of thymocytes from 8-week-oldC3H/HeJ mice to PHA-P). The levels of IL-1 activity in PMXsupernatants were expressed in units per milliliter. One unitwas defined as the amount of IL-1 required to double theproliferative response of C3H/HeJ mouse thymocytes to asubmitogenic concentration of PHA-P (1 p.g/ml).

Assay of IgG Fc receptors on PM4. Immunoglobulin G(IgG) Fc receptors on PMX were measured by a flowcytometric method (27). Human IgGl was purified from theserum of a patient with myeloma and was radioiodinated,and 1251-labeled IgGl binding was quantified by the Crabtreemethod as modified by Guyre et al. (11). Fluorescein isothio-cyanate-labeled IgG1 (fluorescein/protein molar ratio, 3.8)was prepared by the method of Goding (8). For flow cyto-metric analysis, 5 x 106 cells per ml in RPMI 1640 mediumwere-stained for 2 h at 37°C with 4 x 10-' M fluoresceinisothiocyanate-labeled IgGl in the absence or presence of 4x 10-6 M unlabeled IgG (to assess nonsaturable binding). Toexclude nonspecific binding of IgG to the PM4P membrane,PM4) were preincubated with human albumin. PMP werethen washed twice with ice-cold Dulbecco phosphate-buffered saline medium containing 2 mg of bovine serumalbumin per ml, suspended in phosphate-buffered salinemedium containing 2 mg of bovine serum albumin per ml,and analyzed on a cytofluorograph system (Ortho DiagnosticSystems, Inc., Westwood, Mass.) by using 488-nm laserexcitation. To convert the fluorescence values obtained fromthe flow cytometer to the number of molecules of IgG boundper cell, the method of Titus et al. (27) was applied.

Assay of bactericidal activity of PM4. The bactericidalactivity of PM, was tested by a standard method (19).Bacterial suspensions (0.1 ml of Staphylococcus epidermidiscontaining 5 x 107 microorganisms, on average, as definedspectrometrically) were preopsonized with 0.9 ml of poolednormal human serum diluted in Hanks balanced salt solutionwith 0.1% gelatin to a 10% concentration. Incubation wasdone for 30 min at 37°C. The suspensions were centrifuged,and the bacterial pellets were suspended in 1 ml of Hanksbalanced salt solution with 0.1% gelatin (preopsonized bac-teria). One hundred microliters ofpreopsonized bacteria wasmixed with an equal volume of PM4 suspensions (5 x 106cells per ml of Hanks balanced salt solution with 0.1%gelatin) in polypropylene counting vials (Bio-vials; BeckmanInstruments, Inc., Fullerton, Calif.). The bacterium/PMXratio was 10:1. Immediately after the mixtures were pre-pared (time zero) and after 60 min of incubation in a shakingincubator at 37°C, 1.8 ml of sterile water (4°C) was added toduplicate vials and serial 10-fold dilutions in water weremade. Triplicate 20-,ul samples from appropriate dilutionswere plated onto nutrient agar, and bacterial colonies werecounted after 24 h of incubation at 37°C. The results werereported as the percentage of CFU killed (the percentdecrease in the number of CFU after a 60-min incubationperiod). In all experiments, control tests were performed byincubating bacteria without PM+.

Isolation of normal peritoneal fibroblasts. Parietal perito-neum specimens obtained from peritoneal biopsies of CAPDpatients and of women undergoing laparoscopy were mincedand dissociated enzymatically into single-cell suspensions bysequential treatment with 0.4% bacterial collagenase (Wor-thington Diagnostics, Freehold, N.J.) and 0.25% trypsin

E

z

0.05 0.5 5.0 50 CONTROLug MPL/ml

FIG. 1. MPL induction of IFN-y (G-IFN). The values (units permilliliter) presented are the means of at least three determinationsperformed by using cell suspensions obtained from six differenthigh-peritonitis-rate patients ± the standard deviation. Controlvalues were obtained when laparoscopy patient-derived cells wereused. The hatched area represents the range of values obtainedwhen cells from eight CAPD patients without a history of peritonitiswere treated with known PM4) or PLy stimulants and assayedsimilarly.

(GIBCO). Cultures of pure fibroblasts were grown to con-fluence in 35-mm-diameter cluster wells (Costar, Cambridge,Mass.) (4).

Coincubation experiments and normal peritoneal fibroblastproliferation assay. One-hundred-microliter volumes of nor-mal peritoneal fibroblast suspensions and MPL preparationswere added to quadruplicate wells of flat-bottom tissueculture plates (Microtest 1I; Becton Dickinson Labware,Oxnard, Calif.) and cultured for 72 h at 37°C in 5% CO2 in ahumidified atmosphere. One microcurie of [3H]thymidine(Dupont, NEN Research Products, Boston, Mass.) wasadded to each well for the final 6 h of culture. Cultures thenwere harvested on glass fiber filters and assayed for radio-activity by liquid scintillation spectrometry. Normal perito-neal fibroblast proliferation was expressed as the meancounts per minute ± standard deviation ofthe [3H]thymidineincorporated.

Statistics. Results are presented as means ± standarddeviations. Statistical comparisons were made based on arepeated measures analysis of variance or a two-sample ttest using the Welch approximation.

105

Oc.O 0.05 0.5 5.0 50 CONTROL

ug MPL/ml

FIG. 2. MPL induction of IL-2. Other details are as described inthe legend to Fig. 1.

5-

J . CLI N. MICROBIOL .

1

on August 25, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

MPL AND PERITONEAL IMMUNITY 1751

60

17E

40 -

20 ]

O 0.05 0.5 5 50 CONTROLug MPL/ml

FIG. 3. MPL induction of IL-1. Other details are as described inthe legend to Fig. 1.

RESULTSFigures 1 through 5 display the effects of increasing

concentrations of MPL on certain functions of peritonealimmune cells taken from six CAPD patients with histories ofrecurrent peritonitis. The effects ofMPL on the proliferationof peritoneal fibroblasts from these patients are shown inFig. 6. All of the peritoneal immune cell functions studiedwere initially depressed in the CAPD patients when com-pared with the functions of immune cells taken from the 12healthy controls (P < 0.005, by the t test).A repeated measures analysis of variance was performed

to compare the effects of each MPL dosage to the base-linezero dosage (0.2% TEA). Additionally, in order to exclude apossible effect of TEA on the parameters mentioned above,all tests were also carried out by incubating peritoneal cellswithout TEA. Based on a Geisser-Greenhouse-corrected Ftest, a significant effect caused by MPL was found for eachof the five peritoneal immune cell response variables, al-though no significant effect on peritoneal fibroblast prolifer-ation was seen. The presence or absence of TEA had noeffect on the results (data not shown).

Table 1 shows the mean differences in parameters betweencultures incubated without MPL (base line) and the fourMPL dose levels (0.05, 0.5, 5, and 50 ,ug/ml). The corre-sponding P values indicate that a significant differenceexisted for all five of the peritoneal immune cell functionvariables at 0.50 ,ug of MPL per ml or more, while for IL-2production a significant difference occurred at 0.05 tg ofMPL per ml or more.

DISCUSSIONResults of our study demonstrate that the PLy release of

IFN--y and IL-2 in CAPD patients with a high incidence of

30000

10000 a B* I I

ug MPL/ml

FIG. 5. Effect of MPL on PM4 bacterial killing. Results areexpressed as the percentage of bacteria killed. Other details are asdescribed in the legend to Fig. 1.

peritonitis is lower than it is in healthy subjects. Also, thePMf from such patients are less efficient in generating IL-1and in killing S. epidermidis, and they display a reducedmembrane Fc receptor number. These findings are in accordwith the results of previous studies showing that peritonealcells from these patients do not function as well as thoseobtained from patients with a low incidence of peritonitis(15); however, they conflict with those of others (9) whofound the peritoneal immune cell functions in patients un-dergoing CAPD to be near normal, without taking intoaccount the frequency of peritonitis in such patients.We found that PLy and PM4 taken from high-peritonitis-

incidence CAPD patients, when incubated in vitro withincreasing concentrations of MPL, are able to regain asatisfactory ability to release cytokines. This is accompaniedby an increase in the number of PMX Fc receptors and inPMX bactericidal capacity. This effect of MPL on peritonealimmune cell functions has some analogy with that observedin animals and humans seen by incubating PM4X with IFN-y(12, 15, 29).

This MPL-induced activation of PLy and PMX might beable to play an important role in the defense against perito-neal bacterial infections in patients undergoing CAPD, pos-sibly by promoting the killing of intracellular microorgan-isms, which survive in large amounts and for a long time inthe cytoplasm of the PMp of some CAPD patients withrelapsing peritonitis (3, 18).Our results demonstrate that even though MPL has been

found to have some functional analogy with IFN-y (pro-moting macrophage function), it has no direct stimulatory

O 005 0.5 5.0 50 CONTROL

ug MPL/ml

FIG. 4. Effect of MPL on Fc receptor (FcR) density per ceil.Other details are as described in the legend to Fig. 1.

ug MPL/ml

FIG. 6. Effect of MPL on fibroblast proliferation. Results areexpressed as counts of [3H]thymidine incorporated per minute.

VÀ

1

VOL. 27, 1989

TFr

Co

on August 25, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

1752 CAROZZI ET AL.

TABLE 1. Mean response, mean difference from base line, andP values of the peritoneal immune cell functions and

peritoneal fibroblast proliferation in relationshipto in vitro MPL concentrations

MPL Mean difference P

response from base value

IFN-y (U/ml) 0 17.40.05 24.1 6.7 NS"0.50 62.7 45.3 0.00015 97.4 80.0 0.0001

50 83.0 65.6 0.0010

IL-2 (U/ml) 0 0.40.05 0.6 0.2 0.00010.50 1.0 0.6 0.00035 1.2 0.8 0.0001

50 1.0 0.6 0.0114

IL-1 (U/ml) 0 0.30.05 0.4 0.1 NS0.50 1.4 1.1 0.00155 1.6 1.3 0.0001

50 1.3 1.0 0.0002

PM( Fc receptor (103/cell) 0 13.70.05 14.9 1.2 NS0.50 18.1 4.4 0.00745 19.7 6.0 0.0021

50 20.1 6.4 0.0004

Bacterial killing (%, CFU 0 16.2killed in 60 min) 0.05 17.8 1.6 NS

0.50 32.7 16.5 0.00655 45.3 29.1 0.0003

50 43.5 27.3 0.0001

Peritoneal fibroblast prolif- 0 1,208eration ([3H]thymidine 0.05 1,199 -9 NSincorporation [cpm]) 0.50 1,202 -6 NS

5 1,202 -6 NS50 1,200 -8 NS

Zero values are the base l'ne.NS, Not statistically different from the base line.

effect on peritoneal fibroblast proliferation even whenpresent at high concentrations, in contrast to the effects ofIFN-y and IL-1 (22, 28).

In addition, dose-response curves showed that the PLyand PMX production of IFN-y and IL-1-cytokines that aresaid to be involved in the proliferation of peritoneal fibro-blasts in patients undergoing CAPD and, consequently, inthe loss of ultrafiltration capacity (17, 23)-increases, pro-gressively reaching a peak at an MPL concentration of 5p.g/ml. That this cytokine production is lower with MPLconcentrations of 50 p.g/ml than of 5 .ig/ml indicates thepossibility that MPL, in addition to promoting the release ofimmune response-stimulating factors, could also induce therelease of down-regulatory molecules, probably through thecyclooxygenase pathway (10).

Although further in vitro and in vivo studies are necessaryto confirm these results, intraperitoneal MPL therapy maybe considered a potential tool for preventing or treatingperitoneal infections in those patients undergoing CAPDwith abnormalities of peritoneal immune cell functions with-out risking the direct development of peritoneal fibrosis.

LITERATURE CITED1. Boyum, A. 1968. Isolation of mononuclear cells and granulo-

cytes from human blood. Scand. J. Clin. Lab. Invest. 21(Suppl.97):77-109.

2. Brinckerhoff, C. E., and P. M. Guyre. 1985. Increased prolifer-ation of human synovial fibroblasts treated with recombinantimmune interferon. J. Immunol. 134:3142-3146.

3. Carozzi, S., M. G. Nasini, and S. Lamperi. 1988. Cellular andhumoral defense mechanism in peritoneal dialysis, p. 91-95. InG. La Greca, S. Chiaromonte, A. Fabris, M. Feriani, and C.Ronco (ed.), Peritoneal dialysis. Wicthig, Milan.

4. Dayer, J. M., S. M. Krane, R. G. G. Russell, and D. R.Robinson. 1976. Production of collagenase and prostaglandinsby isolated adherent rheumatoid synovial cells. Proc. Natl.Acad. Sci. USA 73:945-950.

5. Dukovich, M., J. M. Severin, S. J. White, S. Yamazaki, and S. B.Mizel. 1986. Stimulation of fibroblast proliferation and prosta-glandin production by purified recombinant murine interleukin1. Clin. Immunol. Immunopathol. 38:381-389.

6. Gery, I., P. Davies, and J. Derr. 1981. Relationship betweenproduction and release of lymphocyte activating factor (inter-leukin-1) by murine macrophages. Cell. Immunol. 64:293-303.

7. Gillis, S., M. M. Ferm, W. Ou, and K. A. Smith. 1978. T-cellgrowth factor: parameters of production and a quantitativemicroassay for activity. J. Immunol. 120:2027-2032.

8. Goding, J. W. 1976. Conjugation of antibodies with fluoro-chromes: modifications to the standard methods. J. Immunol.Methods 13:215-226.

9. Goldstein, C. S., J. S. Bomalaski, and R. B. Zurier. 1984.Analysis of peritoneal macrophages in continuous ambulatoryperitoneal dialysis patients. Kidney Int. 26:733-740.

10. Goodwin, J. S., and D. R. Webb. 1980. Regulation of theimmune-response by prostaglandins. Clin. Immunol. Immu-nopathol. 15:106-111.

11. Guyre, P. M., P. M. Morganelli, and R. Miller. 1983. Recombi-nant immune interferon increases immunoglobulin G Fc recep-tors on cultured human mononuclear phagocytes. J. Clin. In-vest. 72:393-397.

12. Hamburg, S. I., R. E. Manejias, and M. Rabinovitch. 1978.Macrophage activation: increased ingestion of IgG-coatederythrocytes after administration of interferon inducers to mice.J. Exp. Med. 147:593-599.

13. Kaplan, G., D. E. Weinstein, and R. M. Steinman. 1985. Ananalysis of in vitro T-cell responsiveness to lepromatous lep-rosy. J. Exp. Med. 162:917-929.

14. Keane, W. F., and P. K. Peterson. 1984. Host defense mecha-nisms of peritoneal cavity and continuous ambulatory peritonealdialysis. Peritoneal Dial. Bull. 4:122-127.

15. Lamperi, S., and S. Carozzi. 1988. Interferon-gamma (IFN-gamma) as an in vitro enhancing factor of peritoneal macro-phage defective bactericidal activity during continuous ambula-tory peritoneal dialysis (CAPD). Am. J. Kidney Dis. 11:225-230.

16. Lamperi, S., S. Carozzi, A. Icardi, and M. G. Nasini. 1985.Peritoneal membrane host defense mechanism in CAPD. Trans.Am. Soc. Artif. Intern. Organs 31:33-37.

17. Lamperi, S., S. Carozzi, and M. G. Nisini. 1986. Lympho-monokine disorders and peritoneal fibroblast proliferation inCAPD. Trans. Am. Soc. Artif. Intern. Organs 32:35-38.

18. Peterson, P. K., D. Lee, H. J. Suh, M. Devalon, R. D. Nelson,and W. F. Keane. 1986. Intracellular survival of Candida albi-cans in peritoneal macrophages from chronic peritoneal dialysispatients. Am. J. Kidney Dis. 7:146-152.

19. Quie, P. G., J. G. White, and B. Holmes. 1967. In vitrobactericidal capacity of human polymorphonuclear leukocytes:diminished activity in chronic granulomatous disease in child-hood. J. Clin. Invest. 46:668-679.

20. Ribi, E. 1984. Advances in carriers and adjuvants for veterinarybiologics. Iowa State University Press, Ames.

21. Ribi, E. 1984. Beneficial modification of the endotoxin mole-cule. J. Biol. Response Modif. 3:1-9.

22. Schmidt, J. A., S. B. Mizel, D. Cohen, and I. Green. 1982.Interleukin 1, a potential regulator of fibroblast proliferation. J.

J. CLIN. MICROBIOL.

on August 25, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

MPL AND PERITONEAL IMMUNITY

Immunol. 128:2177-2182.23. Shaldon, S., K. M. Koch, E. Quellhorst, and C. A. Dinarello.

1984. Pathogenesis of sclerosing peritonitis in CAPD. Trans.Am. Soc. Artif. Intern. Organs 30:193-194.

24. Takayama, K., N. Qureshi, C. R. H. Raetz, E. Ribi, J. Peterson,J. L. Cantrell, F. C. Pearson, J. Wiggins, and A. G. Johnson.1984. Influence of fine structure of lipid A on Limiilidus amebo-cyte lysate clotting and toxic activities. Infect. Immun. 45:350-355.

25. Takayama, K., N. Qureshi, E. Ribi, and J. Cantreil. 1984.Separation and characterization of toxic and nontoxic forms oflipid A. Rev. Infect. Dis. 6:439-443.

26. Takayama, K., N. Qureshi, E. Ribi, J. L. Cantrell, and K.Amano. 1983. Use of endotoxin in cancer immunotherapy and

characterization of the nontoxic but active lipid A components.ACS Symp. Ser. 231:219-233.

27. Titus, J. A., B. F. Haynes, and C. A. Thomas. 1982. Analysis ofFc-gamma receptors on peripheral blood leukocytes by flowmicrofluorometry. 1. Receptor distribution on monocytes, Tcells and cells labeled with the 3A2 anti-T-cell monoclonalantibody. Eur. J. Immunol. 12:474-479.

28. Trinchieri, G., and B. Perussia. 1985. Immune interferon: a

pleiotropic lymphokine with multiple effects. Immunol. Today6:131-136.

29. Vogel, S. N., and D. L. Rosenstreich. 1979. Defective Fcreceptor-mediated phagocytosis in C3H/HeJ macrophages. 1.Correction by lymphokine-induced stimulation. J. Immunol.132:2842-2846.

VOL. 27, 1989 1753

on August 25, 2020 by guest

http://jcm.asm

.org/D

ownloaded from