Tumor necrosis factor-alpha and interleukin-6 production induced by variations of DR4 polymorphism during the primary mixed lymphocyte reaction.

Serologically defined MHC class II differences provoke release of TNF-alpha and IL-6 during MLR. In order to assess the influence of micropolymorphism defined at the genomic level, we selected informative donors pairs within DR2 DR4 serologically defined unrelated subjects by combining those differing only by DR4 alleles, as assessed by PCR-SSOP (DRB1*0401 to 07). Two groups of MLR combinations were tested including DRB1-identical (group 1, n = 12) and one DRB1 difference (group 2, n = 16). Pairs of HLA-identical siblings (n = 4) and of unrelated subjects differing by two major DR incompatibilities detected by serology (n = 27) were used as controls. We further investigated whether DP and DQ differences contributed to the observed CK production. Comparison of group 2 with group 1 showed that one DRB1 difference had a marked influence on CK production at day 3 (TNF-alpha: 401.8 +/- 85 pg/ml vs. 128.7 +/- 34.5 pg/ml, P = 0.001; SI = 2.97 +/- 0.23 vs. 1.27 +/- 0.09, P < 0.0001; IL-6: 317.6 +/- 44.8 pg/ml vs. 108 +/- 13 pg/ml, P = 0.003; SI = 2.53 +/- 0.37 vs. 1.11 +/- 0.05, P < 0.0001). However, CK release in group 2 was significantly lower than that observed in subjects with two serologically defined DR differences (TNF-alpha: 515.1 +/- 61.4 pg/ml, P = 0.05; SI = 5.61 +/- 0.48, P < 0.0001; IL-6: 545.9 +/- 75.8 pg/ml, P = 0.03; SI = 4.75 +/- 0.58, P < 0.0004). Addition of LPS after one day of MLR resulted in discriminant production of CK in group 2 as compared with group 1. Neither DP nor DQ differences affected CK production. In conclusion, DR subtypic differences induce significant CK release during primary MLR. This in vitro study demonstrates the immunodominance of the DR system in eliciting strong inflammatory mediators release.

Alloactivation induced during mixed-lymphocyte reaction provokes release of tumor necrosis factor alpha and interleukin 6 by macrophages and primes them to lipopolysaccharides.

We tested various factors affecting the production of the CKs IL-6 and TNF-alpha during in vitro alloactivation induced by MLR. Different MLR combinations involving familial and unrelated pairs were evaluated. In family studies, MLRs involving pairs of HLA-identical siblings (n = 6) were characterized by IL-6 and TNF-alpha secretion comparable to the one of autologous controls, in marked contrast with HLA-different combinations (n = 6). These displayed a strong and early (day 3) release of both CKs. In combinations of unrelated individuals involving HLA-A, -B, -C-different but -DR, -DQ-identical pairs (n = 3), low CK release was observed. Addition of LPS (1 micrograms/ml) considerably increased production of IL-6 and TNF-alpha. Clear discrimination of MHC class II differences required a 24-hour preculture followed by addition of LPS for 4 hours, a time relationship compatible with a priming phenomenon due to alloactivation. We conclude that MHC class II alloactivation not only provokes IL-6 and TNF-alpha secretion, but also primes macrophages to LPS so that the production of these CKs is markedly increased and occurs much earlier after LPS addition.

In vitro inhibition of tumour necrosis factor-alpha and interleukin-6 production by intravenous immunoglobulins.

In vitro data about the action of pooled immunoglobulins (Ig) on cytokine (CK) production are controversial. The recent finding of natural antibodies against staphylococcal toxins neutralizing superantigen-induced activation prompted us to design an assay determining their ability to modulate staphylococcal enterotoxin B (SEB) induced CK production (IL-6 and TNF-alpha). Presence of anti-SEB antibodies was demonstrated by a dot-blot assay in the three preparations tested. Preincubation of SEB with pooled Ig prior to addition into the test tube containing PBMCs (neutralizing condition) resulted in a strong inhibition of both TNF-alpha and IL-6 release (TNF alpha: 59 +/- 5% inhibition, P < 0.0001; IL-6: 71 +/- 7% inhibition, P < 0.0001, n = 15). Anti-CD3 MoAb-induced CK production was not modified. During our study it was found that experimental conditions were critical to observe this inhibitory effect. Reversing the previous procedure by adding PBMCs into the test tube containing pooled Ig mixed with SEB resulted in a marked induction of TNF-alpha and IL-6 production. The same observation was made when pooled Ig solely was added (coating condition). F(ab')2 fragments of pooled Ig displayed similar inhibitory capacity when added in neutralizing condition, indicating that the mechanism involved was not Fc dependent. The fragments lost the activating properties of intact Ig when incubated in coating condition, showing that Fc receptor activation occurs in this setting. The present work demonstrates that inhibition of SEB-induced CKs release by pooled Ig can be achieved by SEB neutralization, provided that the experimental conditions avoid activation through the Fc receptor. It can be assumed that similar mechanisms take place in some clinical conditions during which pooled Ig are infused.