Analysis of the relationship between chimerism and the allgeneic humoral response.

BACKGROUND: Persistence of antigens has been suggested to play a role in two opposing immunological phenomena: tolerance and memory. Therefore, we studied the impact of chimerism on alloreactive antibody (allo-Ab) production in kidney transplant patients. METHODS: Thirty-five female renal transplant recipients of male donor organs were classified into the following groups: group 1, 13 sensitized uremic patients on dialysis; group 2, 5 nonsensitized uremic patients on dialysis; group 3, six sensitized patients experiencing graft rejection (3 acute vascular, 1 acute cellular, and 2 chronic); and group 4, 11 nonsensitized with functioning allografts (9 with good function, 1 with acute cellular rejection, and 1 with chronic rejection). Mean duration of dialysis after graft failure was similar in groups 1 (56+/-29.7 months) and 2 (41.8+/-42.4 months), as was dialysis efficiency. Chimerism was measured indirectly in the peripheral blood lymphocytes by polymerase chain reaction amplification of a specific Y chromosome DNA gene sequence with a detection sensitivity limit of 1 male cell per 1 million female cells. Allo-Ab production was measured by the PRA-STAT enzyme-linked immunosorbent assay (Sangstat) method. RESULTS: Chimerism was observed in 60% of groups 1 and 2, 83% of group 3, and 82% of group 4. Among all groups, graft existence, irrespective of its function, positively predicted chimerism in 92% with a sensitivity of 88% and a specificity of 78%. In group 3, all three patients with acute vascular rejection had chimerism and donor-specific allo-Abs. In group 4, eight of the nine patients with no rejection had chimerism. CONCLUSION: Chimerism relates to persistence of allogeneic stimulus irrespective of its function. Chimerism did not confer protection against allo-Ab production or vascular rejection, and its existence was not crucial for sustenance of allo-Ab production.

In vitro IgG1 alloreactive antibody production by circulating memory cells in humans.

This study was designed to investigate the presence of IgG1 alloreactive memory cells in the peripheral blood in humans and their in vitro activation requirements. Alloreactive antibody production was measured after cell activation with cytokines: Interleukin (IL) 2, IL-4 and IL-10, interferon gamma, alloantigens and/or OKT3, lipopolysaccharide or Pokeweed mitogen and Epstein-Barr virus transformation. The examined cells were taken from ten sensitized and five nonsensitized uremic patients with previous graft loss and five normal controls. The titers and percentage panel reactive IgG1 antibody reactivity present in the respective sera was further compared with the in vitro profile. Alloreactive antibody reactivity was measured by PRA-STAT ELISA method. The results show that: 1) Short term control T cell lines or nonsensitized cells were unable to provide the necessary help to autologous B cells to produce alloreactive antibodies of the IgG subclass. 2) Activated cells from sensitized patients produced low levels of alloreactive IgGl antibodies. 3) Stimulation with any of the cytokines and/or mitogens or alloantigens or allopeptides was not sufficient to produce consistent levels of alloreactive IgG 1 antibodies, in spite of its presence in the respective sera.

Fate of recurrent acute interstitial cellular rejection in an HLA identical kidney transplant recipient: impact of donor microchimerism.

We and others have shown that the incidence of acute interstitial rejection in HLA identical and non-identical kidney transplantation is similar. Chronic vascular rejection is, however, rare in full matched recipients. In light of the known correlation between previous acute cellular and chronic vascular rejections, lack of chronic rejection in full matched kidney allografts suggest that the immunological basis of acute and chronic rejections are different and/or only high-grade acute cellular rejection leads to chronic vascular rejection. Herein, we present the case of an HLA identical kidney transplant from a male donor to a female recipient who, because of poor compliance, had frequent acute interstitial cellular rejection culminating into chronic interstitial fibrosis with no evidence of vasculopathy or glomerulopathy characteristic of chronic vascular rejection. Donor cells, as examined by the presence of the Y chromosome DNA, were present in the peripheral blood during the most recent acute rejection but not thereafter. These findings support the notion that acute interstitial cellular rejection can lead to interstitial fibrosis but not chronic vasculopathy/glomerulopathy, and that microchimerism did not confer protection against acute cellular rejection.

Alloantigen-blocking antibodies in sera from highly sensitized uremic patients: antibody class and relationship with lymphocytotoxins.

BACKGROUND: Previously, we identified an antimitogenic IgG antibody separated from sera of patients with known kidney transplant chronic rejection. This antibody inhibits individual patients' own unprimed T helper cell responses to alloantigens as well as a third-party mixed lymphocyte response, but does not inhibit autologous unprimed T helper cell proliferation to adherent anti-CD3 antibody. We suggest that the mechanism of inhibitory action is allogeneic-dependent. METHODS: We used a series of similar experimental designs to test the presence of this antibody in uremic, sensitized patients and have studied its relationship to sensitization as defined by the presence of lymphocytotoxins in four uremic groups: highly sensitized with or without previous graft loss, moderately sensitized with or without graft loss, nonsensitized without previous graft loss, and nonsensitized with graft loss. RESULTS: (1) Sensitization is associated with the presence of a potent antibody that blocks primary mixed lymphocyte response. Primed cells are less susceptible to its antimitogenic action. (2) The blocking antibody activity is present only in sensitized patients who have IgG lymphocytotoxic activity against the same HLA class I antigens. (3) The blocking activity is unequal in the following order: IgG 3 > IgG 1 > IgG 2. (4) Although IgG 1 and 2 fractions contain lymphocytotoxic activity against HLA class I antigens, the IgG 3 fraction does not. CONCLUSIONS: The differential effect of IgG antibodies on naive and memory T cells may explain why humeral responses to alloantigens can be maintained in the presence of blocking antibodies.

Immunoglobulin G subclasses and susceptibility to allosensitization in humans.

Understanding the cellular basis of allosensitization is important because persistence of cytotoxic alloreactive antibodies significantly decreases the chances for receiving a second kidney graft and has a detrimental effect on graft survival rates. In this study, serum levels of IgG subclasses were compared within three groups of uremic patients with different levels of allosensitization (panel reactive antibody level > or = 70%, 10-65% and < or = 10%). All the non-sensitized patients had already lost at least one graft indicating resistance to allosensitization by the previous graft. In addition, the in vitro T-cell proliferation and immunoglobulin M and G subclass production were studied after activation by pokeweed mitogen and alloantigens. The patients' demographics were comparable. The results show that all serum IgG subclass levels in the three groups were comparable and within the range of normal control. Similarly, T-cell proliferation and the in vitro production of IgM was not significantly different. The lymphocytotoxic activity present in each IgG subclass was not associated with an increase in the respective serum subclass level or the in vitro production of the same subclass in the sensitized patients. The data indicate that humoral immunity, as reflected by subclass immunoglobulin levels, is, in fact, normal, in the three groups and that sustenance of cytotoxic antibody production reflects a specific immune response controlled by factors other than intrinsic B-cell abnormality.

Analysis of the in vitro effect of exogenous nitric oxide on human lymphocytes.

We investigated the role of endogenous or exogenous nitric oxide (NO) on human lymphocyte function. We used sodium nitroprusside, nitroglycerine, S-nitroso-N-acetylpenicillamine, sodium nitrite and S-nitroso-L-glutathione as NO-generating compounds. All agents were used at doses that do not produce direct cytotoxicity as measured by trypan blue exclusion as well as chromium-51 release assay. The immune responses examined were peripheral blood lymphocytes (PBL) proliferation and IL-2 production after activation with OKT3 and PHA; allogeneic mediated proliferation and cell mediated cytotoxicity (CML) in MLR; IgG and IgM production after PBL activation with Con-A; proliferation and expression of IFN-gamma and IL-4 mRNA after activation of allogeneic CD4+T cell clones. Cytokine mRNA expression was measured by reverse transcriptase PCR. Our results show that proliferating lymphocytes do not produce a detectable amount of NO as measured by the Griess reaction. In separate experiments, the addition of NG-monomethyl-L-arginine (L-NMMA) did not affect lymphocyte proliferation. Sodium nitroprusside and nitroglycerine exerted a dose dependent antimitogenic effect, inhibited cytokine production and expression, CML generation and antibody production. DNA gel electrophoresis showed no evidence for enhanced programmed cell death. The antimitogenic effect could not be blocked by the NO scavengers, hemoglobin or methylene blue. In contrast, the other nitric oxide generating compounds did not inhibit lymphocyte mitogenesis. The results suggest that human lymphocytes do not produce appreciable amounts of NO to affect lymphocyte mitogenesis. Sodium nitroprusside and nitroglycerine have a potent but nonspecific immunoinhibitory effect on human lymphocyte function by a mechanism other than NO production. In addition, pharmacological levels of NO do not inhibit human lymphocyte mitogenesis.