The longitudinal evaluation of immune responses in patients with periodontal diseases and those undergoing treatment is an important goal but a difficult one because of the lack of appropriate controls. To reduce the variability inherent in many assay systems, we have been evaluating cryopreservation as a way of providing suitable reference data. Ideally, standard preparations of frozen cells would be established at the time of the initial characterization or diagnosis of the disease state and would be included in future studies as treatment progresses.

The effects of cryopreservation on the peripheral blood mononuclear cells of patients with periodontal disease have not been extensively investigated but there have been numerous studies on the immune status of cryopreserved cells of patients with malignant diseases including leukaemia, melanoma, breast and colon carcinoma and sarcoma. Certain immune functions are more susceptible to cryopreservation than others even when well-established freezing and thawing techniques are used showed a selective functional loss of a subset of pokeweed mitogen-responding suppressor T cells after cryopreservation of lymphocytes obtained from normal healthy individuals. We need to clarify whether cryopreservation affects the immunological functions of cells involved in periodontitis before beginning longitudinal studies.

The mechanisms of cytokine production in periodontal infections as well as regulation during Gram-negative infection/sepsis are currently under extensive investigation. Accumulated evidence indicates that the pathology of the periodontal infections is due, in part, to the actions of the host-derived cytokines, induced by lipopolysaccharide. The interaction of lipid A and/or the polysaccharide moiety of lipopolysaccharide with membrane receptors of monocytes results in the synthesis of IL-1?, TNF-?, IL-6, and IL-8. These cytokines not only amplify immune and non-immune cells, but also may cause significant tissue damage by inducing collagenase in fibroblasts and activating osteoclasts.

We have previously reported consistent proliferative responses of peripheral blood mononuclear cells to various stimulatory agents after 2 years in frozen storage. Although the proliferative mechanisms appeared to be intact, the cells’ ability to synthesize and express protein(s) might be altered. Letellier et al. (1991), for example, showed that decreased lymphokine-activated killer-cell activity in lymphocytes after cryopreservation was not associated with a change in the composition of the lymphocyte subsets but with a decreased expression of the CD25 antigen. Alternatively, Friberg et al. (1994) demonstrated that the mean concentration of lipopolysaccharide-stimulated production of TNF-, interferon-? and IL-2 was higher in cryopreserved peripheral blood mononuclear cells than in fresh cultured cells.

This study extends our previous work by evaluating cytokine production by peripheral blood mononuclear cells in response to stimulation with polyclonal B-cell activators, mitogens, superantigens, and bacterial extracts of dental interest after the cells have been stored frozen. We have been unable to trace any studies that have measured cytokines from cryopreserved peripheral blood mononuclear cells after stimulation with periodontal pathogens such as Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans. In addition, we believe this report also describes the first published studies on the stability of cytokine production in cryopreserved mononuclear cells after lymphocyte stimulation with superantigens. This is of particular relevance because of the increased interest in superantigens as causative agents in a variety of systemic diseases and their implication in the aetiology of periodontal disease. The development of procedures for the evaluation of lymphocyte function during periodontitis is in anticipation of longitudinal clinical studies.

Peripheral blood mononuclear cells were obtained from fresh heparinized whole blood and prepared for cryopreservation as previously detailed in Miller et al. (1996). The cells were taken from four individuals with no history of periodontal disease, and a clinically healthy periodontium as defined by generally accepted standards of probing depth, bleeding, and attachment loss. In addition, the donors had not received long-term anti-inflammatory therapy, did not have significant systemic disease, had received no systemic antineoplastic, immunostimulatory or immunosuppressive agents, and had not previously participated in investigations on new drugs or devices within a year of entering our study.

In general, cells were separated by Ficoll–Hypaque density centrifugation, transferred to chilled freezing medium at final concentrations of 10⁷ mononuclears per ml and frozen at a rate of ?1°C/min to a temperature of ?30°C and then at ?5°C/min to a final temperature of ?90°C in a Cryomed liquid-nitrogen programmable freezer (Model 1010; Forma Scientific, Marietta, OH). Frozen samples were stored in the vapour phase over liquid nitrogen (?170°C). For thawing, frozen samples were rapidly transferred to a 37°C water bath followed by sustained agitation and then diluted with chilled defrosting medium. The cell pellet was resuspended in 10 ml RPMI 1640 medium containing 10% A⁺ or AB⁺ serum at 1×10⁶ cells/ml.

These were assessed by phase-contrast microscopy and trypan-blue exclusion. At no time were the concentrations of frozen cells readjusted or altered from those of the original suspension used in the fresh cell assays.

Stimulators included pokeweed mitogen, phytohaemagglutinin P, and concanavalin A (Sigma, St. Louis, MO), and TSST-1 and staphylococcus enterotoxin A (Toxin Technologies, Sarasota, FL). All were prepared from lyophilized preparations by suspension and dilution in RPMI 1640 medium containing 10% human A⁺ or AB⁺ serum to the desired concentration.

Actinobacillus actinomycetemcomitans Y4 was obtained from the collection of the Anaerobe Laboratory of Virginia Polytechnic Institute and State University and a second strain was obtained from the American Type Culture Collection (ATCC 43717) along with P. gingivalis ATCC 33277. Bacteria were grown as previously detailed, harvested, washed extensively, and homogenized in a bead mill (Mini-Bead Beater, Biospec Products, Bartlesville, OK.) with 5-mm dia. glass beads. Protein concentrations were determined by the method of Bradford (BioRad Laboratories, Hercules, CA) and the extracts were frozen until used in culture.

Cells were assayed for blastogenic responses to bacterial preparations, antigens, mitogens, and superantigens in U-bottom microtitre plates. Assays were done in quadruplicate wells containing 2×10⁵ cells and stimulator in a final volume of 200 ?l.

The optimal concentration for each stimulator was determined in preliminary studies (data not shown). Before the beginning of the study, stimulators were diluted in culture medium and stored in portions at ?70°C to prevent degradation by repeated freezing and thawing. Sufficient stimulant and bacterial lysates were prepared to complete the entire study and they were thawed only once. After 3 days’ incubation in a 37°C humidified environment containing 5% CO₂, cells were pulsed with [methyl-³H]thymidine (1 ?Ci per well, 40–60 Ci/mmol). Cells were harvested 24 h later using a PHD multiple automatic sample harvester (Cambridge Technology, Inc., Watertown, MA). Radioactivity was determined in a Beckman LS 6500 liquid-scintillation counter. Thymidine incorporation in unstimulated cells was 3–5% of stimulated cells.