EARLY INDUCTION AND AUGMENTATION OF PARASITIC ANTIGEN-SPECIFIC ANTIBODY-PRODUCING B LYMPHOCYTES IN THE NON-PEYER’S PATCH REGION OF THE SMALL INTESTINE

Ching-Hua Wang¹, Elizabeth M. Richards¹, Robert D. Block², Enio M. Lezcano¹ and Ricardo Gutierrez¹

1 Biology Department, California State university, 5500 University parkway, San Bernardino, CA 92407, USA, ² Department of Neurology and Neural Science, School of Medicine, University of Southern California, Los Angeles, CA 90033, USA

Received 10/12/98 Accepted 10/20/98

3. MATERIALS AND METHODS

3.1. Animals

Male or female SD rats of 6 or more weeks of age were used in all experiments. Rats were purchased from Harlan Sprague Dawley, Inc (Indianapolis, Indiana), housed at the California State University San Bernardino vivarium, and given food and water ad libitum. Animals were randomized into seven groups of three. The pooled control group contained a total of six animals.

3.2.Parasite

Trichinella spiralis was received from Dr. R. G. Bell at the Baker Institute for Animal Health, Cornell University, and maintained by serial passage in retired breeder rats of DA or PVG strains that had been infected with 5,000 muscle larvae at least 30 days prior to use.

3.3. Collection of muscle larvae

The procedures used to isolate the larvae from infected rats and to count intestinal worms have been previously described (26, 27). Briefly, infectious muscle larvae were obtained from minced rat carcasses by pepsin hydrochloride digestion at 37oC for 1 hr. Larvae were isolated by pouring the digested fluid through cheesecloth to remove bones and undigested material and then poured through a 200-mesh sieve, which retained the larvae.

3.4. Preparation of adult antigen

Preparation of the adult antigen of T. spiralis has been previously described (27). Briefly, the intestines were removed from rats three days after an oral infection with 8000 T. spiralis muscle larvae. The collected worms were sonicated to obtain crude larval antigen. A rat monoclonal antibody, 9D4, against T. spiralis antigen (28, 29) was used to affinity purify the 9D4 antigen (29), also called the Ts L1 antigen, which was further conjugated with tetramethylrhodamine isothiocyanate (XRITC, Sigma, St. Louis, MO) fluorescent dye. The 9D4 antigen was first diluted in 0.15 M Nacl and further diluted in the conjugation buffer, 0.5 M, pH 9.5, prepared by mixing 5.8 ml of 5.3% Na₂CO₃ with 10 ml of 4.2% NaHCO₃. The diluted 9D4 antigen was then mixed with XRITC at a ratio of 30 ug of XRITC per mg of protein antigen and the mixture was gently rotated overnight at 4⁰C. The conjugate was dialyzed against 0.15 M NaCl in order to remove the free dye. After several changes, the final preparation was dialyzed in PBS and centrifuged to remove unbound material.

3.5. Infection with muscle larvae

Large numbers of T. spiralis muscle larvae were collected from the pepsin digest previously described. The muscle larvae were flushed from the sieve with 0.85% NaCl then washed again three times with 0.85% NaCl and counted microscopically. A total of 2000 larvae per 1 ml of saline were administered using a feeding tube to rats lightly anesthetized with halothane.

3.6. Obtaining tissues and cryohistological preparation

In order to analyze the isotypes of antibodies produced by the intestinal B lymphocytes and the quantities and tissue distribution of isotype-specific antibody-producing B cells in the intestine, intestinal tissue (15 cm distal to the duodenum), Peyer’s patch (the nearest region to the intestine taken), mesenteric lymph node, and spleen were obtained from normal uninfected rats on days 0 and 5, while the same tissues from rats infected orally with 2,000 Trichinella spiralis muscle larvae (a standard low dose) were collected on days 1, 2, 3, 5, and 7 following infection (early phase infection). The fresh tissue was histologically processed by dipping in cold isopentane (2-Methylbutane, Fisher Chemical, Fisher Scientific, Fair Lawn, NJ) and transferring the dissected tissue to an aluminum foil cup containing Optimum Cutting Temperature (OCT) embedding medium (Miles Scientific, Naperville, IL) and then frozen in liquid nitrogen. Tissue samples were stored at -80oC until processed. Frozen sections of 6 um were cut from the intestine, MLN, spleen, and Peyer’s patch of infected as well as control rats using a Lab Tek Precision Microtome Cryostat (Lab Tek Instruments Co., Westmont, IL), placed on slides coated with poly-L-lysine, and allowed to air dry. Three sections were prepared for each of the tissue samples and then fixed with 2% paraformaldahyde at room temperature for 20 min, washed in PBS pH 7.2 for 5x for 5 min each time.

3.7. Immunofluorescence assay (IFA)

A double labeling immunofluorescence assay was used to reveal the distribution of different B cell populations located within the tissue sections on the slides. The dynamics of the various isotypes of antibodies produced by B cells, specifically IgE, IgA, IgM, IgG1,

IgG2a, IgG2b, and IgG2c, were determined by quantification of the frozen sections using immunofluorescence microscopy. The sections were incubated for 1 hr at room temperature with 1:500 diluted monoclonal mouse anti-rat IgE, IgG1, IgG2a, IgG2b, IgG2c, IgA, and IgM (Zymed Labs Inc., San Francisco, CA). These monoclonal antibodies were H chain specific and in the F(ab)2' form to prevent non-specific binding to the Fc receptors. The primary antibody was aspirated off and the slides were then washed with PBS pH 7.2 three times for 5 min each. Fluorescein (FITC) conjugated goat anti-mouse Ig (F(ab)'₂) and rhodamine (XRITC) conjugated 9D4 T. spiralis antigen were used in a 1:1000 dilution. The FITC-Ig and XRITC-9D4 were mixed 1:1. Both incubations were done by placing 20 ml drops on the sections. Incubation with fluorescent conjugates was done in the dark for 1 hr at room temperature. The slides were then washed again with PBS pH 7.2 three times for 5 min each and mounted in 60% (v/v) glycerine-PBS, or washed a fourth time in dH2O pH 7.2 and mounted with Fluorsave (CalBio Chemical Corp., La Jolla, CA). The conjugated antibodies were visualized using a Nikon optiphot biological microscope with an episcopic-fluorescence attachment EF-D containing a B2A 495nm filter specific for FITC and a G2A 546nm filter specific to XRITC. Double labeled cells in at least 20 villus crypt units (VCU) per intestine, and 20 fields (400x) per section for the spleen, Peyer's patch, and mesenteric lymph node were enumerated and data recorded.

3.8. Statistical analysis

The significance of differences in mean values was first determined by comparing independent means using analysis of variance. Days of infection producing significant F values were further analyzed by Newman-Keul's multiple-range test for each day, as compared to the pooled control and to the previous day of infection. Probability levels less than 0.05 were considered significant.