Mucosal T cells provide helper function but do not proliferate when stimulated by specific antigen in lymphogranuloma venereum proctitis in nonhuman primates

GASTROENTEROLOGY

1988;94:353-66

Mucosal T Cells Provide Helper Function but Do Not Proliferate When Stimulated by Specific Antigen in Lymphogranuloma Venereum Proctitis in Nonhuman Primates MARTIN ZEITZ, THOMAS C. QUINN, ALAN S. GRAEFF, and STEPHEN P. JAMES Mucosal Immunity Immunoregulation, Health, Bethesda,

Section, National Maryland

Laboratory of Clinical Investigation. Institute of Allergy and Infectious

To study antigen-specific immune responses of gutassociated T lymphocytes after gastrointestinal infection, Cynomolgus monkeys were inoculated rectrachomatis of the L2 tally with Chlamydia [lymphogranuloma venereum (LGV)] strain. Infected monkeys developed a chronic proctitis with the appearance of LGV-specific immunoglobulin G-antibodies in the serum. Lymphocytes isolated from the peripheral blood, the spleen, and draining lymph nodes had a vigorous antigen-specific proliferative response to LGV in vitro. Both T and B cells proliferated in response to stimulation with LGV, but B-cell proliferation was T-cell-dependent, as shown by cell separation techniques and cell-cycle analysis with dual-laser flow cytometry. Lymphocytes isolated from both involved and uninvolved lamina propria did not proliferate in response to LGV stimulation, whereas mitogen-induced proliferation was not different in lamina propria lymphocytes and the other lymphocyte populations. This lack of antigen-specific proliferation was not caused by a suppressor effect of mucosal T cells or monocytes or the absence of antigen-presenting cells. In contrast, lamina propria T lymphocytes from infected animals were able to provide antigenspecific help for polyclonal immunoglobulin synthesis by immune B lymphocytes after stimulation with LGV. Thus, in LGV proctitis in monkeys, mucosal antigen-reactive T cells differ from lymphocytes in other sites in that they can provide helper function, but are not able to proliferate in response to LGV antigens. he mucosal immune system is thought to play an important role in host defense against intestinal pathogens. The mechanisms by which this is accom-

T

and Laboratory of Diseases, National Institutes

of

plished involve complex interactions of multiple different types of cells, soluble lymphokines and mediators of inflammation, antibodies, and complement. A central step that controls the specificity of these complex pathways is the recognition of specific antigens by lymphocytes associated with the gastrointestinal tract. The interaction of antigens with lymphocytes activates these cells, causing them to proliferate or carry out multiple different functions. Gut-associated T lymphocytes have been shown to have the potential for important regulatory functions (help, suppression) (1,~) and under certain circumstances to carry out cell-mediated cytolytic functions (3-5). B cells activated in the gastrointestinal tract (a process dependent on T-cell help) have a strong tendency to differentiate into immunoglobulin (Ig) A-secreting cells, which have a predilection to migrate to mucosal surfaces, where they secrete IgA (6,7). Although there is considerable information concerning the details of the secretory IgA response, less is known about the role that T cells play in host defense against pathogens, particularly in the diffuse lamina propria compartment of the gastrointestinal immune system. To further study antigen-specific lymphocyte responses in the gut-associated immune system, we took advantage of a recently developed model of intestinal inflammation and infection in nonhuman

Abbreviations used in this paper: BCG, bacille CalmetteGuBrin: LGV, lymphogranuloma venereum: LPL, intestinal lamina propria lymphocyte; MLN, mesenteric lymph node leukocyte: PBL, peripheral blood lymphocyte; SPL, spleen lymphocyte. fZ 1988 by the American Gastroenterological Association 0016-5085/88/$3.50

GASTROENTEROLOGY

354 ZEITZET AL.

primates.

Rectal

venereum

tis in monkeys rectum

with

mune

responses

terized

the

infection cytes sue

behave

from

LGV-specific

differently

son with

the

lymphocyte

responses

gut-associated systemic

cellular)

study

response

T-cell

the

and

present

immune

trachoma-

inflammation

(humoral

compared from

a lymphogranuloma

a chronic

(8). In the

cellular

to those

with

(LZ) of Chlamydia

causes

and

that

strain

systemic

isolated

found

infection

(LGV)

immune

of the im-

we charac-

to LGV in this of lympholymphoid system.

Vol.Y4,No. 2

(Fc) were obtained from Jackson Immuno Res. (West Grove, Pa.). Bacille Calmette-Gu&in (BCG) Vaccine, U.S.P., was obtained from Glaxo Inc. [Research Triangle Park, NC.), and tuberculin purified protein derivative was obtained from Statens Seruminstitute, Copenhagen, Denmark through Accurate Chemical and Scientific Corp. (Westbury, N.Y.). Tetanus Toxoid, Adsorbed, Purogenated was obtained from Lederle Laboratories (Wayne, N.J.).

tisWe

T lymphocytes stimuli in compariin other lymphoid

Isolation

and

Purification

of Chlamydia

gut-derived

to antigenic populations

sites.

Materials and Methods Animals Cynomolgus monkeys (Macaca fascicularis) of both sexes were imported from the Philippines. They were used after having been quarantined for ~3 mo. For the period of the experiments, animals were kept single in isolation cages. Normal nonhuman primate blood and tissues [Cynomolgus (Macaca fascicularis) and Rhesus (Macaca mulatta) monkeys] were the generous gift of Dr. William London (NINCDS, National Institutes of Health, Bethesda, Md.).

Materials RPM1 1640, fetal calf serum, Hanks’ balanced salt solution without Ca and Mg, phosphate-buffered saline, HEPES buffer, and gentamicin were obtained from Biofluids Inc. (Rockville, Md.). Penicillin, streptomycin, and amphotericin B were obtained from Gibco (Grand Island, N.Y.). Ficoll-Paque solution and Percoll solution were obtained from Pharmacia (Uppsala, Sweden). Ethylenediaminetetraacetic acid, n,L-dithiothreitol, soybean trypsin inhibitor, gelatin, p-nitrophenyl phosphate, I.-leucine methyl ester hydrochloride, indomethacin, 2mercaptoethanol, propidium iodide, ribonuclease A, /-Lchain specific goat-antihuman IgM, y-chain specific goatantihuman IgG, and a-chain specific goat-antihuman IgA conjugated to alkaline phosphatase were obtained from Sigma Chemical Co. (St. Louis, MO.). Deoxyribonuclease I was obtained from Boehringer (Mannheim, F.R.G.). Collagenase was obtained from Worthington Diagnostic Systems, Inc. (Freehold, N.J.). Concanavalin A (con A] was obtained from Miles Scientific, Inc. (Naperville, Ill.). [Methyl-“Hlthymidine and monoclonal antibodies 9.6 (antihuman sheep erythrocyte receptor, anti-Lyt-3, CD2) and anti-B1 (antihuman B-cell, CD20) were obtained from New England Nuclear (Boston, Mass.]. Monoclonal antibodies anti-Leu-2 (CD8, suppressoricytotoxic), anti-Leu-3 (CD4, helper/inducer), and anti-human interleukin 2receptor (CD25) were obtained from Becton Dickinson (Mountain View, Calif.). Purified phytohemagglutinin was obtained from Wellcome Diagnostics (Greenville, N.C.). Purified monkey IgG was obtained from Cappel, Cooper Biomedical Inc. (Malvern, Pa.) and goat-antihuman IgG [F(ab’)J, goat-antihuman IgM (Fc) and goat-antihuman IgA

A human isolate of Chlamydio trachomatis, serovar L2 (LGV) was prepared for inoculation after growth in McCoy cells. Infectivity of the inocula as determined by titration in McCoy cells was 1 x 10 inclusion forming units per milliliter of diluent. For control experimerits, uninfected McCoy cells were processed in the same way as infected McCoy cells to obtain LGV elementary bodies.

Infection of Animals trachomatis

With Chlamydia

Animals were anesthetized with ketamine hydrochloride and preinoculation blood was drawn from the femoral vein. Rectal examinations were performed with a pediatric anoscope and a preinoculation biopsy was done. Monkeys then were infected with 1 ml of the inoculum (1 x lo6 infectious units) by intramucosal injection, and they were kept under isolation conditions for 2 (n = 6) or 6 wk (n = 5). Three monkeys were injected three times with the LGV suspension at weekly intervals and were killed 3 wk after the last LGV injection. Under ketamine hydrochloride anesthesia, a cardiac puncture was performed and animals were killed by bleeding. Before the abdomen was opened, a rectal swab was performed by proctoscopy. The rectum, colon, and small bowel were removed separately after midline abdominal incision. The spleen, mesenteric lymph nodes, and peripheral lymph nodes from the axilla and the inguinal region, as well as local draining lymph nodes from the rectum, were removed.

Enteral Immunization With Bacille Calmette-Gubrin or Tetanus Toxoid Two animals were immunized with BCG by rectal intramucosal injection of 0.1 ml of BCG vaccine as described for the LGV infection. These animals had a negative Tubersol skin test (5 U.S.P. U/test) before this procedure, and both animals developed a positive skin test 4 wk after immunization. Another group of 2 animals was immunized by rectal intramucosal injection of 0.5 ml of adsorbed tetanus toxoid. These animals had negative antitetanus toxoid antibody titers before immunization and developed antitetanus-specific IgG 4 wk after rectal immunization.

Preparation

of Lymphocyte

Suspensions

Because Ficoll-Paque density gradient not uniformly result in sufficient separation

methods do of monkey

February

1988

GUT LYMPHOCYTES IN LGV PKOCTITIS

lymphocytes from whole peripheral blood, a combination of sedimentation with gelatin solutions and on FicollPaque was utilized (9). Briefly, heparinized blood was centrifuged and the plasma was removed. The pellet was resuspended in RPM1 1640 and mixed with an equal volume of 3’3, gelatin solution supplemented with 0.7% NaCl and 0.2% CaCl, ZH,O. The mixture was allowed to stand at room temperature for 1 h. The leukocyte-enriched supernatant was collected and centrifuged, and the pellet was resuspended in RPM1 1640 containing 10% fetal calf serum. The resulting cell suspension was layered on Ficoll-Palque and centrifuged for 25 min at 1000 g. After the interface was collected and washed once with complete medium, the cell suspension was washed once in 30% Percoll solution to decrease the number of contaminating platelets. Spleen lymphocytes (SPLs) and mesenteric lymph node lymphocytes (MLNs) from the different sites were obtained by teasing these tissues and centrifuging on Fllcoll-Paque. Intraepithelial lymphocytes and lymphocytes derived from the intestinal lamina propria (LPLs) were isolated by a modification of a previously described method (3). Approximately 30 cm of the small bowel, the colon without cecum and rectum, and the rectum were washed in phosphate-buffered saline and the mucosa was dissected from the unde.rlying muscular layer with a scalpel. The mucosal pieces were incubated in 100 ml of Ca-free, Mg-free Hanks’ balanced salt solution containing 100 U/ml of penicillin, 100 pgiml of streptomycin, 20 pg/ml of gentamicin, 0.5 pgiml of (amphotericin B, 2 mmol/L of ethylenediaminetetraacetic acid, 25 mmol/L of HEPES buffer, 5 X lo-” mol/L of 2-mercaptoethanol, and 1 mmol/L of u,l.-dithiothreitol in Erlenmeyer flasks on a slowly moving orbital shaker at 37°C for 30 min. The supernatant was discarded and the mucosal tissue was incubated three times in the same medium without r),l.-dithiothreitol for 1 h each time. Intraepithelial lymphocyte-enriched suspensions were obtained as follows: the supernatants were collected, centrifuged, washed in complete medium, and then washed twice in a 30% Percoll solution. The remaining cell suspension was cultured overnight and then centrifuged on Ficoll-Paque. The recovered cells contained ~85% viable lymphocytes. Intestinal lamina propria lymphocyte fractions were obtained by incubating the mucosal fragments in RPM1 l640 containing 10% fetal calf serum, 25 mmol/L HEPES buffer, the antibiotics listed above, 2-mercaptoethanol, 0.01% collagenase, 0.01% deoxyribonuclease, and O.OlI%, soybean trypsin inhibitor at 37°C on an orbital shaker overnight. The suspension was then passed through a stainless steel sieve, and the cells were washed once in c:omplete RPM1 medium without enzymes. The cell pellet was resuspended and washed once in a 30% Percoll solution. The resulting cell preparation was centrifuged on Fic:oll-Paque. The resultant lvmphocyte preparation contained >9b% viable cells. Mitogen-

txnd Antigen-Induced

Proliferation

To consistently detect proliferative responses in the monkey, we found that removal of excess adherent cells was required before assay (data not shown). Plastic

355

adherent cell-depleted lymphocyte suspensions were obtained by incubating cell suspensions at 4 x 10” cells/ml in medium in 100 x 15-mm plastic Petri dishes for 1 h at 37% and then gently removing the nonadherent population with medium at 37°C. In some experiments, plasticadherent cell suspensions were obtained by incubating the Petri dishes with cold medium for 30 min at 4°C. Before addition to lymphocyte cultures, adherent cells were irradiated with 2000 rads (ls7Cs source, Gammator M, Isomedix, Parsippany, N.Y.). Cultures vigorously depleted of adherent cells were obtained by treating the cells at a concentration of 10 X 10” cells/ml with 40 mmol/L of L-leucine methyl ester hydrochloride in medium (pH 7.0) for 30 min at 37°C and washing the cell suspension three times, followed by a 1-h plastic adherence (10). Antigen-pulsing of adherent cells was done by incubating the adherent cells in round-bottom microtiter plates in the presence of LGV elementary bodies for 3 h at 37°C. The cells were washed three times and then irradiated as described before culture. Cell suspensions at a density of 1 x ~10” cells/ml were pipetted into 96-well flat-bottom plates (2 x 10’ cells per well) and mitogens (final concentrations 2.5 pgiml conA or 3 pgiml phytohemagglutinin) or suspensions of the antigen at different dilutions were added. Cultures were set up in triplicate and cultured for 3 days (mitogens) or 5 days [antigens) in a 6% CO,-humidified atmosphere. In some experiments indomethacin at a concentration of 5 PgIml or 0.5 pgiml was added at the initiation of the cultures. Sixteen hours before the termination of the cultures, cells were pulsed with 1 &i(methyl-“Hlthymidine (specific: activity 6.7 Ciimmol) per well. Cells were harvested on filter paper strips using a PHD cell harvester (Cambridge Technology, Inc.). Radioactivity was counted in scintillation vials with 5 ml of scintillation fluid (Ultrafluor) in a Beckman scintillation counter. Separation

of T- and B-Cell

Fractions

T and B lymphocyte-enriched suspensions were obtained by sheep red blood cell separation using neuraminidase-treated sheep red blood c:ells (11). In some experiments. the B cell-enriched fraction was further purified by complement-mediated killing with the monoclonal antibody anti-Lyt-3 (clone 9.6, CD2). In other experiments, mononuclear cell suspensions were depleted of CD&positive cells (suppressoricytotoxic population) by treating the cells with a 1:50 dilution of OKT8-containing ascites for 45 min at 4°C and, after washing the cells, treating with a 1:8 dilution of rabbit complement (low tox H) for 45 min at 37°C. MitogenInduced

and Lymphogranuloma VenereumImmunogJobuJin Synthesis

B lymphocyte-enriched populations from the spleen were cultured with T lymphocyte-enriched suspensions from the different tissue sites at a ratio of 1:l [final cell concentration of 1 X 10” cells/ml) in flat-bottom microtiter plates in the presence of either a 1:lOO dilution of pokeweed mitogen or LGV elementary bodies (3 X 10” infectious units per well) for 10 days in complete medium.

GASTROENTEROLOCY

356 ZEITZET AL.

Supernatants enzyme-linked

were harvested immunosorbent

Enzyme-Linked

and kept assay.

Immunosorbent

LymphogranuIoma

at -20°C

Assay

before

Total

G

M, and

Immunosorbent

lmmunoglobulin Immunoglobulin

Assay

Tetanus

for

Samples of freshly isolated cell suspensions (or thawed cell suspensions) and cell suspensions grown in the presence of conA or LGV in vitro in culture flasks at a density of 1 x 10"cells/ml were stained with the fluoresceinated monoclonal antibodies anti-Leu-2 (CD8) or antiLeu-3 (CD4), or with unconjugated antibodies (anti-Lyt-3 or anti-Bl), using a sandwich technique as described (13). Cells were fixed with l'%, paraformaldehyde and stained for cellular deoxyribonucleic acid by a method similar to that described by Crissman and Steinkamp (14). In brief, cells were resuspended in a Ca-free, Mg-free phosphate buffer containing 6 mmol/L of glucose, and absolute ethano1 was added dropwise under continuous stirring to a final concentration of 70%). Cells were kept in 70%~ethanol down, the cells were for at least 2 h at 4°C. After spinning suspended in buffer containing 1 mgiml ribonuclease A and incubated for 1 h at 37°C. As the final step, cells were iodide. suspended in a 10 pg/ml solution of propidium Cells were analyzed using a dual-laser fluorescenceactivated cell sorter (FACS II, Be&on Dickinson). Data were analyzed with a PDP-10130 computer using programs provided by Dr. T. Chused (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md.].

Statistical

A

lmmunosorbent Assay Toxoid-Specific Immunoglobulin

Staining Acid

for

G, Immunoglobulin

The enzyme-linked immunosorbent assay for total IgG, IgM, and IgA in cuiture supernatants followed the same protocol as described above with the exception that the plates were coated with goat-antihuman IgG, IgM, or IgA antibodies. The concentrations for the coating antibodies were 5 pg/ml for anti-IgG and anti-IgM; because of a low and invariable crossreactivity and the unavailability of antimonkey IgA, plates for the determination of IgA concentrations were coated with 50 pg/ml of antihuman IgA. In parallel, a lo-fold higher concentration of the alkaline phosphatase conjugate of antihuman IgA was used (1:lOOO dilution for anti-IgG or anti-IgM and 1:lOO dilution for anti-IgA). Standards in these assays were monkey IgG or human IgM and IgA.

Enzyme-Linked

No.2

by Dr. L. G. Lum (Fred Hutchinson Cancer Research Center, University of Washington, School of Medicine, Seattle, Wash.) (12).

Phenotypic Analysis and Cellular Deoxyribonucleic

Ninety-six-well flat-bottom plates (Immulon I, Dynatech Lab., Alexandria, Va.) were coated with a suspension of 1 x lo4 elementary bodies (LGV) per ml in 50 mmol/L of sodium carbonate buffer (pH 9.6). Next, plates were washed with phosphate-buffered saline containing 0.05% Tween 20; this was also done after each of the following steps. Nonspecific binding was blocked with 1% bovine serum albumin in phosphate-buffered saline. Serum samples from each animal were tested by incubating 100 ~1 of three different dilutions, each dilution in duplicate, for 2 h at 37°C.One hundred microliters of a 1:lOOO dilution of y-chain specific goat-antihuman IgG conjugated to alkaline phosphatase was added to each well and the plates were incubated for 2 h at 37°C. As the final step, 100 ~1 of a l-mgiml solution of p-nitrophenyl phosphate in lOI)/, diethanolamine buffer (pH 9.8) was added. Color development was quantitated using a spectrophotometer (ImmunoReader NJ-200, InterLab, Thousand Oaks, Calif.). Optical density values were converted into relative units by comparison with a standard serum of a hyperimmunized monkey. Calculations were done using regression analysis after logarithmic transformation of the concentration values. Enzyme-Linked

94,

for

Venereum-Specific

lmmunoglobulin

vO1.

for G

The plaies for determination of tetanus toxoidspecific IgG in the serum were coated with 100 ~1 of tetanus toxoid (Wyeth Laboratories Inc., Marietta, Pa.) at a concentration of 1 flocculation unit/ml. Optical density values were converted into immunoglobulin concentrations by comparison with a standard serum containing a known amount of tetanus toxoid-specific IgG. The reference serum for calculating the tetanus toxoid-specific IgG concentration in the standard serum was kindly provided

Evaluation

Results were expressed as median, minimum, and maximum, and the Kruskal-Wallis nonparametric analysis of variance was performed to compare more than two different groups for significant differences. Two groups were compared by the Wilcoxon-Mann-Whitney test and significant differences were assumed at p < 0.05.

Results Clinical Course of Lymphogranulomo Venereum Infection, Hislopathology, Serologic Response

and

All animals infected per rectum with Chlamydia trachomatis (serovar L2) had evidence of LGV infection by at least one criterion: 13 of 14 animals were culture positive for LGV at the time of death and 13 of 14 animals had anti-LGV-specific IgG as detectable by enzyme-linked immunosorbent assay. None of the investigated animals had detectable anti-LGV IgG antibody titers before infection. Serum antibody concentrations were not significantly different 2 wk (median value 114 relative units) or 6 wk (median value 40 relative units) after infection (compared by the Wilcoxon-Mann-Whitney test). Each of the infected animals developed mild proctitis. Early on (after 2 wk), this was marked by mucosal

1988

February

Table

1.

GLJT LYMPHOCYTES

Concanavalin A-Induced Proliferation of Lymphocyte Infected Rectally With Lymphogranuloma Venereum Control

PBL Median proliferative response (&pm) Range (minimum-maximum) Median background proliferation (cpm)

11

184,050 51.670-325,817 2,145 14

Populations

in Control

IN XI

Monkeys and

PROCTITIS

Monkeys

LGV z wk

P” 203,551

165.440

126,906-165.831 4,755 4

357

199.626-287.076

NS

2.358 5

SPL

Median proliferative response (Acpm) Range [minimum-maximum) Median background proliferation n

138,459 19,625-282.390

(cpm)

MLN Median proliferative response (Acpm) Range (minimum-maximum) Median background proliferation (cpm) n Rectum Median proliferative response (Acpm) Range (minimum--maximum) Median background proliferation (cpm) n

185,085 97.904-230,270

1.931

3.450

7

4

102,776 29,127-108,776

202 .~ 520 99.315s227,sfio .i

194,412

237,617

59,768-308.427

fi8.7OO-291

1,191

2,578

61 3

6

5

4

35,049

151.625 68,857-234,393

987

341

1

NLS

1,250

.B17

NS

101.980 62.248-141.712 890

2

Colon Median proliferative response (Acpm) Range (minimum--maximum) Median background proliferation (cpm) II

38,157-161,425

Small bowel Median proliferative response (Acpm) Range [minimum-maximum) Median background proliferation (cpm) n

56,933-251,812

64,375

96.885 89,147-207.737

600

589

5

3

102.020

162.755 91.540-276.108

68.857 29.944-257.383

NS

597

135.925 134.004-.137.846

836

63 1

1.45A

5

3

3

NS

LGV, lymphogranuloma venereum: MLN, mesenteric lymph node leukocyte; PBL, peripheral blood Iymphoc.vte; SPL. spleen lymphocyte. Lymphocyte populations from the different tissue sites in uninfected controls and monkeys rectally infected with LGV were stimulated in vitro with con A, and [“Hlthymidine incorporation was measured 3 days after stimulation. Values from individual animals represent the median of triplicate samples. The range of triplicate samples was <25%. The median, the minimum. and the maximum of different animals are shown. Background proliferation was not significantly different in the different lymphocyte populations. NS. not significant. ” Lymphocyte populations in the different groups were compared by the Kruskal-Wallis nonparametric analvsis of variance.

erythema associated with increased friability and small aphthous-like lesions. Later (after 6 wk), erythema and friability were absent in most animals, but small (diameter -1-Z mm) nodules with a central superficial lesion were seen and the mucosa had a granulated appearance. Animals did not develop obvious signs of systemic disease. The histopathological changes were characterized by mucosal and submucosal lymphoid follicular infiltrates, partially disrupting the muscularis mucosae. In addition, in some biopsy specimens, ulcerations with chronic inflammation of the underlying submucosa were present and crypt abscesses were frequently observed. In repeatedly infected monkeys, inflammation waz: more intense, but inflammatory lesions remained disc:ontinuous or focal.

Proliferative Populations Antigens

Response of Lymphocyte to Mitogens and CZhlam!~dial

The proliferative capacity of lymphocytes derived from the peripheral blood, spleen, mesenteric lymph nodes, local draining lymph nodes, peripheral lymph nodes, or intestinal lamina propria to con A or chlamydial antigens was tested 2 and 6 wk after rectal infection with LGV and compared with uninfected control animals. As shown in Table 1, the proliferative response to con A (2.5 pgiml) was not significantly different in peripheral blood lymphocyte (PBL), SPL, MLN, or LPL (isolated from either the rectum, colon, or the small bowel) cultures as compared by the Kruskal-Wallis nonparametric

358

GASTROENTEROLOGY

ZEITZ ET AL.

2OOOQ

E 8

a

-

EB

uL LZ-Ag

Spleen

Figure

MY

McCoy

Lymphocytes

responses of spleen lymphocytes from a 1 Proliferative LGV-infected animal (6 wk after infection) to different antigenic stimuli. EB, LGV elementary bodies, 3 X 10” infectious units per well; LZ-Ag, antigen preparation of formalin-killed elementary bodies (serovar Lz], purified by Renografin centrifugation: M@*, antigenpulsed, irradiated plastic adherent cells: McCoy, extract of uninfected McCoy cells, prepared by the same procedure as used for LGV elementary bodies. [“Hlthymidine incorporation (S cpm) was measured 5 days after exposure to the different stimuli and the median of triplicate samples is shown. The range of tht: triplicate samples was
analysis of variance. No significant differences were found between infected or uninfected animals. In addition, lymphocyte populations enriched with intraepithelial lymphocytes (obtained from the intestinal incubations in Ca-free, Mg-free Hanks’ balanced salt solution before the digestion procedure) were isolated and purified from 2 infected animals and 2 controls. Concanavalin A-induced proliferative response expressed as the change in counts per minute of the intraepithelial lymphocyte-containing fraction ranged between 46,125 and 258,635 (median 87,707; n = 4) and thus was comparable to that found in the LPLs. Next, proliferative responses of different lymphocyte populations after antigenic stimulation with suspensions of Chlamydia trachomatis elementary bodies were measured. As detailed in the Methods section, these studies were carried out by culturing lymphocytes with whole LGV elementary bodies for 5 days in microtiter plates. There was a clear dosedependent proliferative response of lymphocytes isolated from peripheral blood, spleen, or lymph nodes from infected animals, with a maximum response at a dose of 3 x lo4 infectious units per well. Concentrations higher than 25 x lo4 infectious units per well were inhibitory (data not shown); thus, all

Vol. 94, No. 2

of the following experiments were performed at the optimal concentration of 3 X 10” infectious units per well. The time dependence of the LGV-induced proliferative responses was also tested: at day 2 there was a detectable response, which plateaued between days 4 and 6 and decreased at day 8 (data not shown); therefore, if not stated otherwise, all cultures were terminated at day 5 after antigenic stimulus. In addition, when lymphocytes from infected animals were co-cultured with autologous, LGVpulsed, irradiated plastic adherent cells, [“Hlthymidine incorporation was comparable to direct stimulation with chlamydial antigens (Figure 1). Antigen specificity of this proliferative response was shown in several ways: first, no proliferation was observed in response to uninfected McCoy cell preparations (Figure 1). Second, lymphocytes from infected animals did not proliferate in response to a control antigen to which they had not been exposed 1 pgiml). Finally, when lympho(tetanus toxoid, cytes derived from the peripheral blood, the spleen, and mesenteric lymph nodes in uninfected animals. were stimulated with the same antigenic preparation (3 x 10” infectious units of LGV elementary bodies), a significant proliferative response was found in only 2 of 15 uninfected, healthy monkeys (change in counts per minute of 19,724 and 16,488). In human studies it has been shown that a high percentage of otherwise healthy persons has an antigen-specific proliferative T-cell response to LGV antigens, indicating natural contact with Chlamydia trachomatis (15). It is therefore likely that the LGV-induced proliferation seen in 2 uninfected monkeys was due to natural contact with Chlamydia trachomatis or other cross-reactive antigens. In the other 13 control monkeys, proliferative responses after LGV stimulation ranged between 0 and 6857 (change in counts per minute, medium 457) (shown in Table 2 for the different lymphocyte populations). Table 2 shows the combined data of the proliferative response of lymphocytes derived from the peripheral blood, spleen, mesenteric lymph nodes, the lamina propria, and the intraepithelial compartment in LGV-infected animals. Peripheral blood lymphocytes, SPLs, and MLNs showed a clear proliferative response after in vitro stimulation with LGV elementary bodies. None of the lymphocyte populations derived from the intestinal lamina propria from either infected rectum or other sites (colon, small bowel) or from suspensions enriched with intraepithelial lymphocytes responded with proliferation to chlamydial antigens, despite the fact that these cell populations were shown to respond to con A in the same experiment. Comparing the proliferative responses 2 wk and 6 wk after infection, there was a significant increase in the response of SPLs but not

GUT LYMPHOCYTES

Table

2.

Lymphogranuloma Lymphogranuloma

Venereum-Induced Venereum-Infected

Proliferation of Different Lymphocyte Populations in Monkeys 2 and 6 Weeks After Infection and in Uninfected Control

PBL Median proliferative response (Acpm] Range [minimum-maximum] Median background proliferation (cpm] n SPL Median proliferative response (Acpm) Range (minimum-maximum) Median background proliferation (cpm) n MLN Median proliferative response (Acpm) Range (minimum-maximum] Median #background proliferation (cpm) n PLN Median iproliferative response (Acpm) Range [minimum-maximum) Median background proliferation (cpm) n KLN Median proliferative response (Acpm) Range (minimum-maximum) Median background proliferation (cpm] n Rectum Median proliferative response (Acpm] Range (minimum-maximum] Median background proliferation (cpm] n Colon Median proliferative response (Acpm) Range (minimum-maximum] Median background proliferation (cpm) n Small bowel Median proliferative response (Acpm) Range (minimum-maximum] Median background proliferation (cpm) n IEL Median proliferative response (Acpm] Range (minimum-maximum] Median background proliferation (cpm] n

IN LGV PROCTITIS

1,909

o-6.857 2.595 11

2,104 744-19.724” 1,621 5

1,055 205-16,488b 1,009 5

ND

LGV 2 wk

19,015 3,235-45,629

6,858

5

5

20,273 8.228-24,606 3,929

Controls P”

27.485 14.657~50,369

7,404

NS

42,635 9.959-57,368

p < 0.05

3.541

4

8,556 1,156-30,506 2,154

4,888 1.707-15.647

5,883 2.374 2

61.742

NS

921

5

4,111-7,655

ND

LGV 6 wk

359

5

6,61Y 3,771-9,468 2,149 r,

NS

32,988 27.703-32.988

10,781

1,688

1

0

261 206-317

5,465 1

0 2,265 3

0

1,056

139

200

O-478

O-400

509

462

4

3

8 1,296

3

3

0

ND

-

1,205

3

O-16 965

0 -

0 -

1,935

300 O-350

602 2

-

1,634 2

IEL, intraepithelial lymphocytes; MLN, mesenteric lymph node lymphocytes; ND, not determined; PBL, peripheral blood lymphocytes; PLN, peripheral lymph node (axilla); RLN, local draining lymph node from the rectum; SPL, spleen lymphocytes. Lymphocyte populations from the different tissue sites in uninfected controls and monkeys rectally infected with lymphogranuloma venereum (LGV) were stimulated in vitro with LGV elementary bodies, and [3H]thymidine incorporation was measured 5 days after stimulation. Values from individual animals represent the median of triplicate samples. The range of triplicate samples was <30%. The median, the minimum, and the maximum of different animals are shown. Background proliferation was not significantly different in the different lymphocyte populations. ’ Lymphocyte proliferative responses 2 and 6 wk after infection were compared by the Wilcoxon U-test: NS, not significant. ” In 2 of 15 animals a significant proliferative response to stimulation with chlamydial antigens was observed. Maximum [“Hlthymidine incorporation in the other 13 uninfected animals was 6857 cpm (median 457, range O-6857).

360

GAS’I’KOI-:NTE:KOI,O(;Y

ZEITZ ET AL.

Figure 2. Lymphogranuloma

venereum-induc:ed proliferative response of different lymphocyte subpopulations obtained from spleen lymphocytes from a I.CV-infected animal. SPL, unseparated spleen lymphocytes (background, lfi54 cpm); SPL-T. SRBC-rosetting population (background 903 cpm); +M@, adding increasing numbers of irradiated adherent cells to SPI,-T; SPL-B. non-T cell fraction (CD2-positive cells <8%. background 753 cpm): B Lyt 3. non-T cell fraction treated with Lpt 3 and complement (CD2-positive cells --Ccl/,,, background 1522 cpm). [“H]thymidine incorporation (A cpm) was measured 5 days after exposure to an optimal dose of LGV elementary bodies. The median of triplicate samples (range < 2tl’g1) is shown.

of PBLs or MLNs at 6 wk. At the latter time point, responses of SPLs were significantly higher than responses of MLNs. In 2 animals 2 wk after infection and in 2 animals 6 wk after infection, lymphocytes from peripheral lymph nodes in the axilla and local draining nodes from either the rectum or the inguinal region were obtained. Proliferative responses to chlamydial antigens in vitro were substantially higher in lymphocytes derived from local draining nodes compared with lymphocytes isolated from peripheral or mesenteric lymph nodes (Table 2). [“HlThymidine incorporation was comparable in lymphocytes from peripheral nodes and mesenteric nodes (Table 2).

Phenotypic Characterization Proliferating in Response to Lymphogranuloma Venereum

of Lymphocytes

The LGV-responsive cells in responding populations were characterized by cell separation techniques. In those studies, T cells separated by sheep red blood cell rosetting techniques [the percentage of anti-B1 (CD20, anti-B cell)-positive cells was ~4%) in the rosetting population] responded with [“HIthymidine incorporation after LGV stimulation, and this response was enhanced by adding adherent cells. B cell-enriched populations separated by the same technique (the percentage of CD2-positive cells

1’01. 94. No. 2

was <8($, in the nonrosetting population) had a poor response, which was even lower after further depletion of T cells by treatment of the original B-cell preparation with anti-sheep red blood cell receptor monoclonal antibody and complement (Figure 2): in the latter population, the percentage of CD2-positive cells was <3%. The LGV-responsive cells in responding populations were also characterized in unseparated cell populations with the use of correlated deoxyribonucleic acid and surface immunofluorescence flow cytometry. In these studies stimulated lymphocytes were stained with different monoclonal antibodies specific for T or B cells and with propidium iodide to stain cellular deoxyribonucleic acid. The increase in red fluorescence is a quantitative measurement of the deoxyribonucleic acid content in single cells and thus allows for the separation of cells in the (;l phase. S phase, or G2IM phase of the cell cycle. Parallel staining with fluoresceinated monoclonal antibodies specific for cell surface antigens on lymphocytes makes it possible to distinguish individual cell subpopulations entering the cell cycle after different stimuli. As shown in the fluorescenceactivated cell sorter analysis depicted in Figure 3, both T and B cells moved into the S phase and G2iM phase of the cell cycle after LGV stimulation, whereas after co11 A stimulation only ‘1‘cells entered the cell cycle. The percentage of cells entering the S phase and G2 phase after LGV stimulation was higher for T cells compared with B cells (10.3% and 6.3%, respectively). L,ymphogranulorrla venereuminduced proliferation of CD4-positive (helper/in ducer subpopulation) and CD8-positive lymphocytes (suppressoricytotoxic subpopulation) was also determined by this method: here, although proliferation of both subpopulations was observed, more CD&positive cells responded than (:D&positive cells (CD4, S + G2: 6.8% t 2.2; CDs. S t G2: 2.R%, + 0.5; median ? range of two experiments). Finally, using this method it was shown that ,X)(X, of the cells in the S phase and G2 phase of the cell cycle were interleukin 2 receptor-positive. Together. the data on the proliferation of separated lymphocyte populations shown in Figure 2 and the cell cycle analysis data shown in Figure 8 indicate that LGVinduced proliferation is due to both T- and B-cell proliferation in unseparated whole cell cultures, but B-cell proliferation is dependent on the presence of T cells. Luck of I~yrnpf~ogrt~nulo~~~u Venereum-Specific Proliferation Proprio Lymphocytes

of Lamino

The failure of lymphocytes derived intestinal lamina propria to respond with

from the prolifera-

February

GUT LYMPHOCYTES

1988

IN KV

PROCTITIS

361

64 A

%

C

0

0

0

46 -

F

P [r 46 -

32 -

16 -

I

I

10

I

100

I 1000

10

100

1000

I

I

10

100

1000

GREEN FLOURESCENCE Figure

3. Fluorescence-activated

cell sorter profile of mitogen (con A)- or LGV-stimulated spleen lymphocytes. Spleen lvmphocytes isolated from a LGV-infected animal were stimulated with either con A (upper panels) or LGV (lower panels). After 3 days lymphocytes were stained with propidium iodide (red fluorescence. vertical axis] and either control fluorescein isothiocyanate stain (A and D), fluorescein isothiocyanate antisheep erythrocyte receptor monoclonal antibody (ClJ2), or fluorescein isothiocyanate anti-B1 (CDZO). Most con A-stimulated lymphocytes that have increased deoxyribonucleic: acid content are CD&positive (B), whereas LGV-stimulated lymphocytes with increased deoxyribonucleic acid content are both CD2-positive and (X)2-negative (E). In contrast, very few con A-stimulated lymphocytes having increased deoxyribonucleic acid content are CD20-positive (C). but both CD20-positive and CD20-negative lymphocvtes have increased deoxyribonucleic: acid content (F).

tion to chlamydial antigens might have been due to absence of functional antigen-presenting cells, or the presence of suppressor T cells or inhibiting monocytes. These possibilities were investigated by the following experiments. First, it was shown by flow cytometry that freshly isolated LPLs contained 8% monocytes before plastic adherence and 5% monocytes after plastic adherence. In comparison, freshly isolated PBLs contained 13% monocytes before plastic adherence and 4% monocytes after plastic adherence. In addition, when unseparated LPL cultures or isolated T cells were supplemented with increasing numbers of autologous, irradiated plasticadherent cells and cultured in the presence of LGV at optimal concentrations, no proliferative response was observed above background proliferation, whereas spleen T-cell thymidine incorporation was increased after addition of adherent cells (Figure 4). Thus, lack of LGV-induced LPL proliferation was not due to lack of an antigen-presenting cell population.

Second, when LPL cell suspensions were depleted of CD&positive lymphocytes (suppressoricytotoxic) by complement-mediated lysis with OKT8, these depleted cell populations were also unresponsive to LGV stimulation (data not shown]. In addition, when co-cultures were performed of nonresponding LPLs with responding autologous lymphocytes from mesenteric lymph nodes at different ratios, no suppressive effect of LPLs on MLN proliferation was observed (Figure 5). These studies indicate that the presence of suppressor T cells did not account for the lack of LGV-induced proliferation. Third, LPLs or SPLs were vigorously depleted of monocytes by treatment with I.-leucine methyl ester hydrochloride and plastic adherence. After this treatment, monocytes were undetectable by flow cytometry, and antigen-induced proliferation was reduced by 75% in SPLs, but could be restored to the level of untreated cultures by adding 5% or ‘10% irradiated adherent cells from the spleen. However, no signif-

362

GASTKOENTEROLOGY

ZEITZ ET AL.

Mitogen- and Lymphogranuloma VenereumInduced Polyclonal Immunoglobulin Synthesis In Vitro

20

n

0

15 -

x

5 lo5-

1 Figure

Vol. 94, No. 2

SPL-T

4. Lymphogranuloma venereum-induced proliferative responses of T lymphocytes isolated from either spleen lymphocytes (SPL-T) or intestinal lamina propria lymphocytes from the colon (LPL-T) from a monkey with LGV proctitis. Irradiated autologous plastic adherent cells (MQ) from the peripheral blood were added in increasing numbers. [‘Hlthymidine incorporation (A cpm) was measured 5 days after exposure to an optimal dose of LGV elementary bodies. The median of triplicate samples is shown; the range in the different groups was <18%. One representative experiment of three performed is shown. Addition of antigen-presenting cells does not result in a proliferative response of lamina propria T cells to antigenic stimulation with LGV.

icant proliferative response was observed when LPLs were depleted of monocytes with I.-leucine methyl ester and accessory cells from spleen were added. In addition, culturing in the presence of different concentrations of indomethacin (5 &ml and 0.5 pgiml) also did not result in a proliferative response of LPLs to LGV stimulation. Thus, the failure of LPLs to proliferate in response to LGV stimulation was not due to an inhibitory effect of intestinal monocytes. Experiments were also carried out to prove that the failure of intestinal lamina propria lymphocytes to proliferate in response to LGV stimulation was not an artifact of the method of isolation of intestinal lymphocytes. Peripheral blood and spleen lymphocytes from an LGV-infected animal (6 wk) were subjected to the collagenase digestion protocol, and there was no significant change in either background 3465 cpm proliferation (median 3709 cpm untreated; collagenase-treated), con A-induced proliferation (median 247,513 cpm untreated; 227,055 cpm collagenase-treated), or LGV-stimulated proliferation (median 29,133 cpm untreated; 27,961 cpm collagenase-treated). Thus, the failure of lamina propria lymphocytes from LGV infected animals to proliferate in response to LGV stimulation is not due to inhibition of the capacity of lymphocytes to respond to mitogens or antigens because of the method of isolation of the lymphocytes.

B-lymphocyte-enriched populations obtained by sheep red blood cell rosetting (~8% CD2-positive cells) from the spleens of monkeys infected three times with LGV or from control monkeys were co-cultured with T lymphocytes [sheep red blood cell-rosetting cells, ~4% anti-B1 (CD20)-positive cells] from the peripheral blood, the spleen, lymph nodes, or the lamina propria in the presence of either pokeweed mitogen or LGV elementary bodies. Total IgG and IgM were measured in culture supernatants. As shown in Table 3, T cells from preinfection peripheral blood lymphocytes, T cells from the different tissue sites after infection, and T lymphocytes from noninfected control monkeys were able to provide help for polyclonal immunoglobulin synthesis after pokeweed mitogen stimulation to a comparable degree. However, after culturing in the presence of LGV, preinoculation peripheral blood T lymphocytes and T cells from control animals did not augment IgG or IgM synthesis significantly above background values, whereas T lymphocytes from the different sources after infection manifested significant helper function. In particular, T lymphocytes from the intestinal lamina propria from both colon and small bowel, which did not proliferate in re-

60c

Figure

5. Autologous co-cultures of lymphocytes isolated from mesenteric lymph nodes (MLN) or the intestinal lamina propria (LPL) from a LGV-infected animal. Intestinal lamina propria lymphocytes that did not respond with proliferation to stimulation with LGV in vitro were added to MLNs that did respond to LGV with proliferation in increasing proportions: the cell number per well was held constant (Z X 10” cells/well). The cocultures were grown for 5 days in the presence of an optimal dose of LGV, and [:‘H]thymidine incorporation was measured. The median of triplicate samples is shown; the range in the different groups was <15"A,. One representative experiment of three performed is shown. The observed decrease in deoxyribonucleic acid synthesis in the co-cultures was not different from that expected.

2

+ 97

+ 147

-’ 187

+ 45

243 2 86

2,870

182 k 27

1,489

499

1,432

1,017 _f 418 404 t 134

None

Mitogen-

-t 462

2 82

+ 922

k 354

+ 259

2 228 ? 638

500 -+ 105

3,126

1,006

10,041

1,683

1,681

11,537 4,869

PBLpre

7,453

7,706

2,603

23,994

1,593

2 1,385

403

4,792

t

22

+ 1,152

i

r

ND

+ 111

ND 2 62

2 51

+ 890

3,448

14,974 + 69

+ 94

606 k 61

7,463

9,381

8,961

+ 547 -t 886

7,641

SPL

t

ND

192

L 720

2 882 ND

RLN

to spleen

C and

+ 20

2 3,102

2,537

6,445

882

6,944

3,050

8,032

3,401

t

206

2 564

k 53

+ 361

+ 332

+ 115

+ 201

t- 829

Colon

9,058

B cells

Immunoglobulin

6,295

946

ND ND

ND ND

+ 1,017

2

2 76

6,907

2 427

6,381 4,607

SmB

M Synthesis”

1,473

14,059

ND ND

ND ND

ND

t

ND 273

+- 1320

sPL*

?

1228

2 77

2 37

k 282

ND

2 160

ND

266 2 16

542

1,136

5,624

1,671

9,876

colon*

lamina propria T lymphocytes from noninfected control animals: LGV, LGV-stimulated PBLpre, preinfection peripheral blood T lymphocytes; PWM, pokeweed mitogen-stimulated the rectum; SmB, lamina propria T lymphocytes from the small bowel; SPL, spleen T concentrations in culture supernatants; values are given in nanograms per milliliter and the isolated from the spleens of lymphogranuloma venereum (LGV)-infected animals were cononhuman primates. Cultures were stimulated with either pokeweed mitogen or LGV cultures. Lamina propria T cells from infected animals provide helper function for IgG and

4,061

24,475

560 + 50

4,581

10,113

13,113

T cells added

lmmunoglobulin

6,318

Venereurn-induced

PBLpost

29,474

and Lymphogranuloma

Colon, lamina propria T lymphocytes from the colon not including the rectum; Colon*. cultures; ND, not determined; PBLpost, postinfection peripheral blood T lymphocytes; cultures (positive control); RLN, T lymphocytes isolated from draining lymph nodes from lymphocytes; SPL*, spleen T lymphocytes from noninfected control animals. ” IgG or IgM median I range of two separate samples is shown. B-lymphocyte-enriched cell suspensions cultured with T cells isolated from different tissues from uninfected and LGV-infected elementary bodies, and total IgG and IgM were measured 10 days after initiation of the IgM synthesis by spleen B cells after antigenic stimulation with LGV elementary bodies.

fgG fgM

fgG fgM LGV

Experiment PWM

fgG fgM

fgG fgM LGV

1

3. Pokeweed

Experiment PWM

Table

364

Table

GASTROENTEROLOGY

ZEITZ ET AL.

4.

Mitogen- and Antigen-Induced Monkeys

Rectally

Immunized

Proliferation with Bacille [“H]Thymidine

PBLpre Experiment Con A PPD

1

Experiment Con A PPD

2

of Lymphocyte Calmette-Gukrin incorporation

PBLpost

Populations

(change

in counts

From

Different

Tissue

Sites in 2

per minute) SmB

Colon

MLN

SPL

Vol. 94, No. 2

182,340 + 44,750 34 -t 867

299,950 21,191

t 17,893 t 4,261

163,995 12,659

+ 29,140 + 595

313,018 3,980

k 26,610 2 1,478

271,517 2 1,540 333 + 579

111.741

-+ 19,147 0

t 18,490 0

275,409 48,933

t 19,875 + 2,239

216,876 11,527

k 9,145 -+ 6,233

244,111 2,325

t 10,767 t 174

121,062

+ 4,886 0

234,142

+ 2,390 0

214,975

Colon, lamina propria lymphocytes from the colon (including the rectum); MLN, lymphocytes isolated from mesenteric lymph nodes: PBLpost, postimmunization peripheral blood lymphocytes; PBLpre, preimmunization peripheral blood lymphocytes; SmB, lamina propria lymphocytes from the small bowel; SPL, spleen lymphocytes. Concanavalin A (con A)- and tuberculin purified protein derivative (PPD)-induced proliferation in the different lymphocyte populations from 2 animals was measured 6 wk after rectal immunization with bacille Calmette-GuBrin by [“Hlthymidine incorporation. Values are given as median and range from triplicate samples. Background proliferation was not significantly different in the different populations (median: 3,523 cpm).

sponse to LGV-stimulation, did provide help for immunoglobulin synthesis by B cells after antigenic stimulation with LGV. No experiments could be performed with rectal T cells because the numbers of LPLs obtained from the rectum were insufficient for these experiments. It was also shown that, corresponding to the results obtained by measuring IgG and IgM, spleen and lamina propria T cells from immunized animals were able to provide help for IgA synthesis by spleen B cells after stimulation with both specific antigen (LGV) or pokeweed mitogen, but the concentrations of IgA were significantly lower compared with IgG and IgM (pokeweed mitogen: B cells alone, 30.2 -C 6.2 ngiml; + SPL T cells, 79.2 & 4.2 ngiml; + LPL T cells, 127.2 +- 39.2 ngiml; LGV: B cells alone, 27.1 ? 8.4 ngiml; + SPL T cells, 99.1 ? 13.4 ng/ml; + LPL T cells, 116.7 -t 22.4 ngiml. Results from experiment 1 as shown in Table 3; the median and range of two separate samples is given). The lower values obtained for IgA could be due at least in part to the low precursor frequency of IgA B cells in the spleen. In animals inoculated once with LGV, a helper effect of T cells from either the intestinal mucosa or other tissue sites was also observed, but was less pronounced (data not shown). Taking B-lymphocyte-enriched populations from normal, noninfected animals and culturing those cells with T lymphocytes from different sources from infected or uninfected animals in the presence of LGV antigen, there was no significant increase of IgG synthesis above background values with nonim(1.5 to mune T cells, and only a small increase a-fold) with T lymphocytes from infected animals (background value, 561 +- 162 ng/ml IgG; +T cells from infected animals, 948 ? 351 ngiml IgG; median 2 range, n = 3).

Immunization

With

Other

Antigens

To investigate if the lack of antigen-specific proliferation of lamina propria-derived lymphocytes was specific for LGV proctitis, normal nonhuman primates were also immunized with either rectal intramucosal injection of tetanus toxoid or BCG, and proliferative responses of lymphocytes from the different tissues were measured after stimulation with specific antigens (tetanus toxoid or tuberculin purified protein derivative). Rectal immunization with tetanus toxoid did induce antitetanus toxoid-specific serum IgG antibodies [no detectable antitetanus toxoid IgG before immunization, 4 wk after immunization 0.85 pgiml and 4.79 PgIml, n = 2), but PBLs from neither of the 2 immunized animals responded with proliferation to different doses of tetanus toxoid in vitro (data not shown); thus these animals were not further investigated. Rectal immunization with BCG, however, induced a vigorous proliferative response of PBLs to stimulation with tuberculin purified protein derivative in vitro. Table 4 shows the combined data of 2 nonhuman primates immunized by rectal injection with BCG. Again, LPLs from both the colon and small bowel did not respond with [“Hlthymidine incorporation to antigenic stimulation in vitro, whereas lymphocytes from other tissue sites (PBLs, SPLs, and MLNs) had a high proliferative response in the presence of tuberculin purified protein derivative. Antigen specificity of this response was indicated by a lack of proliferation of preimmunization PBLs (Table 4).

Discussion mates rectal

Rectal infection with LGV in nonhuman priclosely resembles the human disease (8). After LGV infection, monkeys develop a proctitis

Februar\! I ~188

histolo,gically characterized by a focal discontinuous inflammation, with the appearance of mucosal lymphoid aggregates, crypt abscesses, and occasional giant cell granulomas. This infection provides an opportunity to determine the nature of antigenspecific lymphocytes in various mucosal compartments after challenge of the mucosal immune system with this common pathogen. The results of this study show that challenge of the mucosal immune system with LGV results in a humoral immune response to LGV and in the appearance of LGVreactive lymphocytes (as detected by LGV-induced proliferative responses) in organized lymphoid tissues, such as draining lymph nodes or the spleen, and in the peripheral blood. The proliferative responses occurred among the lymphocyte subpopulations that would be expected to generate anti-LGV antibody responses (CD4-positive T lymphocytes and B lymphocytes). In contrast to previous published observations (16), we found that LGV-induced proliferation was antigen-specific and that both T and B cells proliferate after LGV stimulation. Similar results have been obta,ned previously in both humans and different animal models of chlamydial infections (15,17-19). Ilnexpectedly, such LGV-reactive lymphocytes were not present in the intestinal lamina propria of the rectum, the site of infection, or in other lamina propria sites of the bowel. This was not due to a global inability of lamina propria lymphocytes to proliferate, because mitrogen-induced proliferation was not different in LPLs compared with the other populations. The lack of antigen-specific proliferation was not caused by suppressor T cells or inhibiting monocytes in the lamina propria nor by an inadequate antigen-presenting cell population. In addition, experiments in monkeys immunized rectally with BCG showed that lymphocytes from the intestinal lamina propria also did not proliferate to antigen [tuberculin purified protein derivative) in the presence of a vigorous antigen-specific proliferative response of lymphocytes from other sources. In view of these data it might at first be concluded that antigen-specific lymphocytes are absent in the lamina propria during LGV infection. This is not the case, however, as it was shown that LGV-specific T cells are present in the intestinal lamina propria if such cells are sought in an assay of antigen-specific effector function. Thus, lamina propria T cells cultured with LGV were able to provide help for B cells in an assay of polyclonal immunoglobulin production. It should be noted that although the product of this res,ponsc was nonspecific, the T-cell help was specific, inasmuch as preimmune peripheral blood T cells or nonimmune control T cells from different sources were not able to provide help when stimu-

GIJT LYMPHOCYTES

IN LCV PROCTITIS

365

lated with LGV. Thus, after LGV challenge of the mucosal immune system, antigen-reactive T cells are present in the gut mucosa that can provide helper function but are not able to proliferate to antigenic stimulation. On this basis it is reasonable to conclude that the antigen-specific T cells in the lamina propria in LGV proctitis are differentiated helper effector cells that do not respond with proliferation upon antigenic stimulation. The lack of proliferation of antigen-reactive cells in the gut mucosa might be caused by the specific conditions in the microenvironment of the lamina propria or by a different state of responsiveness of lamina propria T cells. Evidence that certain lymphokines may alter the responsiveness and function of antigen-reactive cells is given by two recent studies, in which it has been shown that cloned T lymphocytes become unresponsive to antigen with regard to proliferation after exposure to interleukin-2 but at the same time develop effector functions (20,21). As lamina propria lymphocytes are able to produce high amounts of interleukin-2 after mitogenic stimulation (22 and Zeitz M et al., unpublished observations], high concentrations of interleukin-2 or other lymphokines, or both, in the environment of the intestinal mucosa may induce an unresponsive state of antigen-specific T lymphocytes to proliferative stimuli. In addition, it has recently been shown that antigenic stimulation may not result in proliferation of certain cloned T lymphocytes or T-cell hybridomas, but may induce lymphokine production (23,24). Although T-cell clones and hybridomas may be different in their signal transduction after stimulation compared with T lymphocytes in vivo, these studies indicate that dissociation of proliferative responses and lymphokine production can occur after prior antigen or lymphokine exposure. That antigen-specific T cells in the intestinal lamina propria exhibit helper-effector function rather than proliferate upon challenge with antigen fits well with recent phenotypic data regarding this population. It has been demonstrated that a subpopulation of CD4-positive cells that does not express the Leu-8 antigen or the 2H4 antigen provides virtually all helper activity in pokewoed mitogenstimulated cultures (25-27). The great majority of CD4-positive LPL T cells has this phenotype (CD4positive, Leu-8-negative, 2H4-negative) (.13). Thus, the major function of CD4-positive T cells in the lamina propria rnay be to provide helper function. The data presented here allow one to postulate the sequence of cellular events that follows mucosal infection with Chlamydiu trachomutis. At the initiation of rectal infection, T cells encounter LGV antigens in organized mucosal lympboid tissue. This primary interaction leads to the proliferation of

366

ZEITZ ET AL

antigen-specific T cells in lymphoid follicles and particularly in draining nodes. The cells then enter the circulation and migrate to the gut mucosa, at which point they may have undergone differentiation events that render them no longer capable of proliferative responses to LGV antigens. Thus, upon secondary contact with LGV in the lamina propria the cells do not respond with proliferation, but rather by providing help for Ig synthesis. This sequence of events is consistent with the hypothesis that T cells in the lamina propria are a more differentiated population of lymphocytes that mediate specific effector functions upon encountering antigens.

References 1. James SP, Fiocchi C, Graeff AS, Strober W. Immunoregulatory function of lamina propria T cells in Crohn’s disease. Gastroenterology 1985;88:1143-50. 2. Elson CO, Machelski E, Weiserbs DB. T cell-B cell regulation in the intestinal lamina propria in Crohn’s disease. Gastroenterology 1985;89:321-7. 3. James SP, Graeff AS. Spontaneous and lymphokine induced cytotoxic activity of monkey intestinal lymphocytes. Cell Immunol 1985;93:387-97. 4. Targan S, Britvan L, Kendall R, Vimadalal S, Sol1 A. Isolation of spontaneous and interferon inducible natural killer like cells from human colonic mucosa: lysis of lymphoid and autologous epithelial target cells. Clin Exp Immunol 1983; 54:12-22. 5. Gibson PR, Jewel1 DP. Local immune mechanisms in inflammatory bowel disease and colorectal carcinoma. Gastroenterology 1986;90:12-9. 6. McDermott M, Bienenstock J. Evidence for a common mucosal immunological system. I. Migration of B lymphoblasts j Immunol into intestinal, respiratory and genital tissues. 1979;122:1892-8, 7. McWilliams M, Phillips-Quagliata JM, Lamm ME. Characteristics of mesenteric lymph node cells homing to gut associated lymphoid tissue in syngeneic mice. J Immunol 1975; 115:54-8. 8. Quinn TC, Taylor HR, Schachter J. Experimental proctitis by rectal infection with Chlamydia trachomatis in non-human primates. J Infect Dis 1986;154:833-41, M, Fujiwara T, Hayami M. Blastogenic response of 9. Tatsumi peripheral blood lymphocytes to mitogens in cynomolgus monkeys (Macaca fascicuiaris). Am J Primatol 1982;3:111-9. M, Lipsky PE. Phenotype of the acces10. Thiele DL, Kurosaka sory cell necessary for mitogen-stimulated T and B cell responses in human peripheral blood: delineation by its sensitivity to the lysosomotropic agent, I.-leucine methyl ester. J Immunol 1983;131:2282-90. 11. Weiner MS, Bianco C, Nussenzweig V. Enhanced binding of neuraminidase-treated sheep erythrocytes to human T lymphocytes. Blood 1973;42:939-46. 12. Lum LG, Culbertson NJ. The induction of suppression of in vitro IgG anti-tetanus toxoid antibody synthesis by human lymphocytes stimulated with tetanus toxoid in the absence of in vivo booster immunization. J lmmunol 1985:135:185-91. 13. lames SP, Fiocchi C, Graeff AS, Strober W. Phenotypic analysis of lamina propria lymphocytes: predominance of helperinducer and cytolytic T-cell phenotypes and deficiency of suppressor-inducer phenotypes in Crohn’s disease and control patients. Gastroenterology 1986;91:1483-9.

GASTROENTEROLOGY

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JA. Rapid stimulation measurement 14. Crissman HA, Steinkamp of DNA, protein, and cell volume in single cells from large mammalian cell populations. J Cell Biol 1973;59:766-71. 15. Qvigstad E. Skaug K, Thorsby E. Proliferative human T cell responses to Chlamydia trachomatis in vitro. Acta Path01 Microbial Immunol Stand [C] 1983;91:203-9. 16 Bard J, Levitt D. Chlamydia trochomatis stimulates human peripheral blood B lymphocytes to proliferate and secrete polyclonal immunoglobulins in vitro. Infect Immunol 1984; 43:84-92. 17 Brunham RC, Martin DH, Kuo CC, et al. Cellular immune response during uncomplicated genital infection with Chlamydia trachomatis in humans. Infect lmmunol 1981; 34:98-104. 18. Qvigstad E. Digranes S, Thorsby E. Antigen-specific proliferative human T-lymphocyte clones with specificity for Chlumydia trcchomatis. Stand J Immunol 1983:18:291-7. in chlamydial eye 19. Young E, Taylor HR. Immune mechanisms infection: cellular immune responses in chronic and acute disease. J Infect Dis 1984;150:745-51, 20. Otten G, Wilde DB, Prystowsky MB. et al. Cloned helper T lymphocytes exposed to interleukin 2 become unresponsive to antigen and concanavalin A but not calcium ionophore and phorbol ester. Eur J Immunol 1986;16:217-25. L, Kagnoff MF. A murine cytotoxic ‘1 21. Klein JR, Lefrancois lymphocyte clone from the intestinal mucosa that is antigen specific for proliferation and displays broadly reactive inducible cytotoxic activity. J Immunol 1985;135:3697-703, 22. Fiocchi C. Hilfiker ML, Youngman KR. et al. Interleukin 2 activity of human intestinal mononuclear cells. Decreased levels in inflammatory bowel disease. Gastroenterology 1984;86:734-42. 23. Ashwell JD, Cunningham RE, Noguchi PD. Hernandez 1). Cell growth cycle block of T cell hybridomas upon stimulation with antigen. J Exp Med 1987:165:173-94. 24. Harris DT. Kozumbo W]. Cerutti P, Cerottini C. Molecular mechanisms involved in T cell activation. 1. Evidence for independent signal-transducing pathways in lymphokine production vs. proliferation in cloned cytotoxic T lymphocytes. J lmmunol 1987;138:600-5. 25. Kansas GS, Wood GS, Fishwild Dbl. Engleman EG. Functional characterization of human T lymphocyte subsets distinguished by monoclonal anti-Leu-8. J Immunol 1985; 13432995%3002. 26. Morimoto C. Letvin NL, Distaso JA. Aldrich WR, Schlossman SF. The isolation and characterization of the human suppressor induced T cell subset. J Immunol 1985:134:1508-15. 27. Damle NK. Mohagheghpour N. Engleman EG. Soluble antigen-primed inducer T cells activate antigen-specific suppressor T cells in the absence of antigen-pulsed accessory cells: phenotypic definition of suppressor-inducer and suppressoreffector cells. J Immunol 1984;132:644-50.

Received November 7. 1986. Accepted September 17. 1987. Address requests for reprints to: Dr. Martin Zeitz, National Institutes of Health, NIAID, Building 10, Room llN250, Bethesda, Maryland 20892. M. Zsitz is supported by grant Ze 188/&l from the Deutsche Forschungsgemeinschaft. The authors thank Linette Edison for expert technical assistance in performing the studies 011 the fluorescence-activated cell sorter and Dr. William London, NINCDS. National Institutes of Health. for providing normal monkey tissues. The authors also thank Dr. Warren Strober for emouragement and critical review of the manuscript.